MXPA96003127A - Bacterioc - Google Patents

Abstract

The present invention relates to strains of Micrococcus varians that produce a composition having a bactericidal activity, including deposited strains CNCM I 1586 and CNCM I 1587 and biologically pure cultures thereof, and also including strains having the nucleotide sequence SEQ ID NO: 2 include nucleotides 154 to 228 of SEQ ID NO: 2, and which may also include the amino acid sequence SEQ ID NO: 1, ainoacid sequence SEQ ID NO: 3. An isolated peptide, which in a microorganism, and particularly in MIcrococcus varians, is a signal peptide that initiates the expression of a bactericidal composition. Fragments of nucleotides that encode a bacteriocin composition derived from Micrococcus varians and to prepare compositions to obtain the bacteriocin and to obtain recombinant microorganisms. The nucleotide fragments include, in particular, nucleotides 154 to 228 and nucleotides 88 to 153 and combinations thereof, nucleotides 88 to 228, identified in the sequence lists.

Description

BACTERIOCINE The present invention relates to a bacteriocin, a strain producing this bacteriocin, a process for preparing this bacteriocin and the use of this bacteriocin and / or of a producer strain of this bacteriocin, in the manufacture of foodstuffs and cosmetic products. .

KfiTADO DK THE TECHNIQUE: Bacteriocins were isolated in many bacteria, Gram positive and Gram negative. They are molecules of an essentially protein nature that have a bactericidal power and, due to this fact, provoke an antagonistic reaction between the bacterium that produces it and one or several bacterial species. In addition, the spectrum of inhibition of bacteriocins, is limited most of the time to the species close to the species of the bacterium that produces it. Above all, some bacteriocins have been found in lactic acid bacteria. For example, EP 0643136 (Société des Produi ts Neetlé) describes the identification of two bacteriocins of Streptococcus termophilus. Likewise, a bacteriocin was isolated from Lactococcus lactis (App. And Env.Microbium, 58, 279-284, 1992. J. of Bio.Chem. 268, 16361-16368, 1993). But to date, no bacteriocin from Micrococcus varians is known. On the other hand, Mlcrococcus varians was used a lot in the food field, on?) All in the fermentation of the meat to make salchichonería products, such as salamis and sausages, for example. Therefore, it would be very useful to have a strain producing a bacteriocin, in order to fight against pathogens. The object of the present invention is that of responding to this need.

SUMMARY OF THE INVENTION For this purpose, the bacteriocin according to The present invention is a bacteriocin from Micrococcus variane having the amino acid sequence SEQ ID NO: 1 or any amino acid sequence that differ from the sequence SBQ ID NO: 1 by a substitution, a deletion and / or an insertion of 1 to 4 amino acids In addition, any nucleotide fragment coding for this bacteriocin, especially the core-thymic fragment having the sequence SEQ ID NO: 2, also falls within the scope of the present invention. Likewise, the strain according to the present invention is a strain of Micrococcus varians that produces this bacteriocin, especially the strains Micrococcus varians CNCM 1-1586 and CNCM 1-1587. In the process for preparing the bacteriocin according to the present invention, a strain of Micrococcus varians that produces the bacteriocin, especially the strain CNCM 1-1586 or the strain CNCM 1-1587, in order to obtain a culture medium containing 107 1011 germs of that strain for me, centrifuge the culture that was obtained, and then prepare an extract of what supernatant that bacteriocin contains. Finally, the use of the bacteriocin Micrococcus varians according to the invention, comprises the use of its nucleotide sequence, as well as its signal sequence, and the use of the supernatant extract containing the bacteriocin and of a Micrococcus varians strain producing that bacteriocin for the preparation of food products and cosmetic products.

DETAILED DESCRIPTION OF THE INVENTION: In the following description, the bacteriocin according to the present invention will be referred to as "variacin". In the sense of the present invention, an arbitrary unit (ua) is defined as the inverse of the rate of the largest dilution at which a sample still teaches a bactericidal power in the test known to the man of the trade, under the designation of "agar well _ Q test ", which literally means test of the well drilled in the agar In the sense of the present invention, the term" fragment "or" DNA fragment "must be understood as a DNA fragment of single or double coding cape j_5 partially or totally, and which can be synthesized, reproduced in vi tro for example with the known method called "Polymerase chain Reaction", or reproduced in vivo in a bacterium of the type Escherichia coli, for example. For the purposes of the present invention, "homologous fragment" means any fragment that differs from the fragments according to the invention, only by the substitution, elimination or insertion of a small number of bases. In this framework, it will be considered in Particularly as homologs, two DNA fragments that, due to the degeneracy of the genetic code, code for the same polypeptide. It will also be considered as a homologous fragment, that which has more than 80% homology with the fragment according to the invention. In the latter case, the homology is determined by the relationship between the number of bases of a homologous fragment and that of a fragment according to the invention. Finally, in the sense of the present invention, "hybrid fragment" means any fragment that can hybridize to the fragments according to the present invention, with the Southern-Blot method (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, US A., 1989, chapters 9.31 to 9.58). Preferably the hybridization is conducted under stringent or stringent conditions, in order to avoid non-specific or unstable hybridizations. It was isolated from strains CNCM 1-1586 and CNCM 1-1587, a protein factor, in this case a bacteriocin, which has a potent bactericidal power. It was called variacin, to this bacteriocin of Micrococcus varians that presents, therefore, the sequence of amino acids SEQ ID NO: l that is described in the list of the sequences below. Considering the interest presented by the variacin, the invention also relates to any bacteriocin having a sequence of amino acids that differ from the sequence SEQ ID NO: 1, by a substitution, a deletion and / or an insertion of 1 to 4 amino acids. Indeed, that bacteriocin, which has an amino acid sequence that differs from the sequence SEQ ID NO: 1 by a substitution, a deletion and / or an insertion of 1 to 4 amino acids, can have an inhibition spectrum at a. type or a bacterial species broader than that of the variacin mentioned, for example. It was also possible to select from the two strains CNCM 1-1586 and CNCM 1-1587, a chromosomal fragment coding for the variacin according to the invention. That fragment has the sequence SEQ ID NO: 2 which is given in the list of the sequences below. In view of the interest presented by the present invention, the invention also relates to any nucleotide fragment coding for the variacin according to the present invention, especially the nucleotide fragments homologous or hybridizing with the sequence SEQ ID NO: 2. In particular, the invention relates to nucleotides 88 to 153 of the sequence SEQ ID NO: 2 coding for the variance signal peptide, nucleotides 154 to 228 of the sequence SEQ ID NO: 2 coding for the variant excreted according to the present invention and / or nucleotides 88 to 228 of the sequence SEQ ID NO: 2 encoding the bacteriocin fused to its signal peptide. The coding fragment for the excreted variacin can be advantageously used to express the variacin according to the present invention, in a plant or in a microorganism other than Micrococcus varians. For this purpose, nucleotides 154 to 228 of the sequence SEQ ID NO: 2 can be cloned into an expression vector in front of a promoter and a signal sequence, and behind a ternate, while respecting the framework of reading, and then you can introduce that vector into a plant, a bacterium or a yeast, in order to increase its inhibition spectrum to certain bacteria, for example. The signal sequence of the invention can be used, by fusing nucleotides 88 to 153 of the sequence SEQ ID NO: 2 to a gene of interest, while respecting the reading frame, and then cloning the set into an expression vector of Micrococcus variane, in order to allow the expression and excretion of the protein encoded by the aforementioned gene of interest in Micrococcus variane, for example.

Nucleotides 88 to 228 of the sequence SEQ ID NO: 2 can be cloned into an expression vector of Micrococcue variane and introduce it into another strain of Micrococcue variane, so that the latter produces the variacin according to the present invention. In addition, the Micrococcue variane strain comprising, integrated in its genome or by means of an expression vector, a DNA fragment coding for the. variant according to the invention, is also an object of the present invention. Especially the strains of Micrococcus varians, deposited on June 7, 1995, according to the Budapest Treaty, in the National Collection of Cultures of Microorganisms, PASTEUR INSTITUTE, 25 rué du Docteur Roux, F-75724 PARIS CEDEX 15, France, in where they received the deposit numbers CNCM 1-1586 and CNCM 1-1587, form the object of the present invention. The Micrococcue variane are Gram positive bacteria, catalase positive, aerobic that are immobile permanently. They have a spherical shape and are presented in the form of irregularly arranged tetrads. The colonies of Micrococcue variane are yellow on BHI medium. The optimum growth temperature of these strains is 25 ° C / 37 ° C.

The strains CNCM 1-1586 and CNCM 1-1587, which form the subject of the present invention, both metabolize glucose and fructose. The strain CNCM 1-1587 also metabolizes sucrose and furanose. In addition, strain CNCM 1-1586 contains two plasmids of 4 and 12 kb, while strain CNCM 1-1587 has a single 7 kb. The supernatant of the culture of the strains CNCM 1-1586 and CNCM 1-1587, present a spectrum of inhibition to the growth of other relatively large bacteria. Among the bacteria sensitive to these supernatants, it can be understood Lactococcue lactie, Lactobacillus helveticue, Lactobacillus delbrueckii eubep. bulgaricus, Lactobacillus delbrueckii eubep lactie, Lactobacillus delbruecki subep. delbureckii, Lactobacillus johneonii, Lactobacillus plantarum, Lactobacillus eake, Lactobacillue curvatue, Leuconoetoc carnoeu, Leuconoetoc mesenteroidee eubep. meeenteroidee, Streptococcue termoiphilue, Listeria monocí togenee, Enterococcue faecalie eubep. faecalie, the spores and vegetative cells of Bacillue subtilis, Bacillus cereue, Bacillue polymyxa, Bacillue circulane, Bacillue pumulue and Bacillus licheniformie and the Cloetridia, for example.

Preferably, at the time of the procedure to prepare the variacin, a strain of Micrococcue variane producing said bacteriocin is grown in medium or under favorable growth conditions, in order to obtain a culture medium containing 107 to 1011 germs of that strain by me, the culture that was obtained is centrifuged, and then an extract of what supernatant that bacteriocin contains is prepared. To produce this extract, the strain of Micrococcue varians producing the variacin, according to the present invention, especially the strain CNCM 1-1586 or the strain CNCM 1-1587, can be grown in a medium and in conditions favorable to the growth of Micrococcue varians . For this purpose, it can be cultivated especially in the BHI medium, at 25 ° C / 37 ° C in aerobiosis, under agitation, until obtaining a concentration of 107 to 1011 germs per ml of medium, for example. The normal culture, obtained in this way, is centrifuged at 4000-6000 g and the extract of the supernatant which contains the aforementioned bacteriocin is collected. The present invention also relates to the use of variacin, especially in the form of extract, or the use of a strain of Micrococcue variane producer of this bacteriocin to prepare food products and cosmetic products. It can be used in particular in culture, one of those strains of Micrococcue variane, according to the present invention, in the fermentation of the meat to prepare room I, in order to fight against contamination by Cloetridia, for example. The variacin can be used in the form of raw or purified extract, added to the yeast obtained from bacteria resistant to that variacin in the preparation of cheeses, especially cheeses of the ozzarella type, to avoid the holes produced by Bacillue polymixa whose spores survive fermentation, and of the bovine type, to fight against contamination by Lieteria onocytogenee, for example. The variacin can be used in the form of crude or purified extract, or one of the two strains, as an additive or active agent against the pathogenic bacteria in the preparation of dessert creams, such as pasteurized custards, in order to fight against contamination by Cloetridia, Bacillue cereue, or bacterial strains such as Lieteria, for example. In addition, variacin can be used in the form of crude or purified extract, or one of the two strains, as an additive or active agent against pathogenic bacteria at the time of the preparation of cosmetic products, such as rehydrating creams, or deodorants, of way to fight against the pathogenic bacteria of the skin, for example. The variacin according to the present invention is characterized in more detail below with the help of different microbiological, biochemical and genetic data that illustrate its properties. The percentages are given by weight. 1Ü Unit of antibacterial activity. "? Sar ffell-Test": In the context of the present description, the bactericidal power is defined in terms of arbitrary units.What supernatant of a classic Micro-ib coccue culture varies according to the present invention, which is prepared for example under the conditions illustrated in Example 1, it typically exhibits an activity of 640 ua / ml.Also, a supernatant concentrate that was prepared, for example under the conditions illustrated in Example 2, typically presents a With the aid of the aforementioned Well-Teet Agar, it is determined if a sample still exhibits a bactericidal power at a given dilution rate 25 To do this, in a Petri dish, they are inoculated 15 ml of medium MRS agar containing an indicator strain at a rate of 105-106 CFU / ml Indicator strain is used as a strain sensitive to variacin, in this case the strain Lactobacillus bulgarus (YL 5) or the strain Lactobacillus hel veticue (N2), for example. 5 mm diameter holes are drilled in the culture medium. The samples of the supernatant or the concentrate of the supernatant that is going to be tested are poured into the holes, at a rate of 50 μl per hole. It is preincubated for 2 hours at 4 ° c and then incubated at 30 ° C or 37 ° C, overnight, according to the indicator strain used. Following the incubation, the indicator strain grew and halos of inhibition appear. The j_5 rate of dilution at which a sample no longer presents bactericidal power, is the dilution rate from which the inhibition zone is no longer distinguished.

Inactivation by enzymes: With the help of the said Well-Teet Agar, it is determined whether the variacin isolated according to the present invention is inactivated in the presence of a proteolytic enzyme or in the presence of catalase. For this, 5 mg / ml of enzyme is added to a Concentrate the supernatant of the culture illustrated in Example 2. The enzyme is allowed to act at the incubation temperature for 60 minutes before depositing the sample in the Well-Teet Agar well. At the same time, a control is made, which is prepared with the same concentrate at a pH of 7 and without adding enzyme, it is incubated at 37 ° C for 60 minutes, before depositing it in the well of Agar Well -Teet, in order to compare the halos of inhibition in the presence of enzyme with the halo of the control. The diameter of the inhibition halo of the control is 27 mm. Table I below presents the results obtained with the enzymes that were tested with the help of the indicator strain, Lactobacillus helveticue (N 2). In that table, as in Table II, the enzyme is designated by its type, the name of the supplier and the item number of the supplier. The figure 0 means that there is no halo, in other words that the bactericidal power of the variacin was compromised by the incubation with the enzyme. The figure 27 indicates that there is still a halo of 27 mm corresponding to the full activity of the bactericidal power of variacin. i? BH? J Inactivation Temperature Enzyme incubation (mm) (° C) Catalase (SIGMA C-10) 30 27 Pronaze (SIGMA P-8038) 37 0 Proteinase K (MERCK 1000 144) 37 0 Ficin (SIGMA F-3266) 37 0 Table II below presents the results obtained with the enzymes tested with the help of the indicator strain, Lactobacillus bulgarue (7L5).

TABLE II Inactivation Temperature Enzyme incubation (mm) (° C) Catalase (SIGMA C-10) 30 27 Pronaze (SIGMA P-8038) 37 0 Proteinase K (MERCK 1000 144) 37 0 Ficin (SIGMA F-3266) 37 0 The results presented in tables I and II show that all proteolytic enzymes suppress the bactericidal power of variacin. These results highlight the fact that the variacin is of a protein nature and that this protein part is involved in the bactericidal power. The fact that no influence of catalase is observed on the bactericidal power of variacin, also demonstrates that the growth inhibition of the two indicator strains is not due to the antibacterial activity of H202 which is known to have an activity similar to that of bacteriocins, since H202 had also been degraded by catalase.

Spectrum of inhibition of the u supernate of the crop? TW km _-_______ mw tm___? U? __ _m ^ * With the help of the Agar Well -Teet, it is determined if the bactericidal power of the supernatant of the culture containing the variacin according to the present invention, presents an inhibitory activity of the growth of the different strains of spores and bacteria. In this way, the spectrum of inhibition of what is supernatant is determined. To carry out these tests, 15 ml of a classical medium inoculated with 15 μl of a culture of the strain to be tested and which is prepared during the preceding night is poured into the Petri dishes in order to obtain a bacterial concentration of 105 to 106 per ml of normal medium. The classical medium is the medium favorable to the growth of the strain to be tested. On the other hand, when the strain to be treated should grow from spores, it is inoculated with 105 to 106 spores, per my coating medium. A 5 mm diameter hole is drilled per Petri dish. A 50 μl sample of the supernatant of the culture illustrated in Example 1 is deposited therein. It is preincubated at 4 ° C for 2 hours and then incubated at a temperature favorable to the growth of the strain that was tested. for the time necessary to coat the plate with a visible bacterial lawn. The effect or degree of inhibition is characterized by the diameter of the inhibition halo that is observed. It is considered that the inhibition is very strong (++++) if the halo has a diameter of 18-28 mm, strong (+++) for a diameter of 10-17 mm, medium (++) for a diameter of 5-9 mm, small (+) for a diameter of 1-4 mm and void (-) if not observe no halo. In this way, 32 strains of lactic bacteria of different species and sub-species are tested and it is found that each one is resistant to what is supernatant. The detail of the results of these tests is presented in Table III below. In this table III, as in the following tables, the designation or number of the strain indicated is the number attributed to it in the Neetlé collection (Address: NESTEC, SA, Research Center, vere-chez-lee-Blanc , CH-1000 Lausanne 26, Switzerland). The indicated temperature is the incubation temperature during the test. The medium indicated is the normal medium favorable to the growth of the strain to be tested. ip TTT TABLE J.5 twenty In this table III, it is noted that the spectrum of inhibition of what is supernatant is broad in the sense that for the different species that were tested, the degree of inhibition is homogeneous. Likewise, it is considered that the spectrum of inhibition of the supernatant of a culture that produces the variacin of the invention, is broad in the sense that it is not limited to lactic species, but extends to other species of Gram-positive bacteria , above all to the undesirable or pathogenic bacteria Lieteria innocua, Lieteria welhía, Lis teria onocytogenes, and to the spores of the Bacilli, for example, as they show, the results presented in table IV below. twenty TABLE IV TABLE IV The results illustrated in this table IV allow, among other things, to provide interesting uses of the supernatant or the purified variant, as an additive in the preparation of food products as an active agent against pathogenic bacteria, especially in meat products. against Cloetridia, in cheeses, against Lieteria monocytogenes, in the dessert creams, against Bacillue cereue and Listeria, or in fresh pasta or sauces for fresh pasta, against bacilli, products where the preceding strains have precisely their origin, for example. Finally, the variacin does not exert any effect of inhibiting the growth of Gram-negative bacteria, as can be seen by seeing the results illustrated in Table V below.

TABLE V Species No. T (° C) Means Inhibition E. coli 1 30 BHI - Enterobacter chloacae 72 30 BHI - Salmonella ana tum XIV / 20 30 BHI - • Salmonella typhimurium XIV / 274 30 BHI - Peeudo onae fluorescens 3 30 BHI -Spectrum is inhibition of concentrated concentrate containing Yarialna: Proceed in the manner described above, except for the fact that the effect of inhibition of the growth of the different strains of spores and bacteria produced by a concentrate of what supernatant was obtained as described in example 2. The same species and sub-species are tested as in the above. The results of these tests are presented in Tables VI, VII and VII below. The designation or strain number indicated is the number attributed to it in the Neetlé collection (Address: NESTEC, A.A., Research Center, vere-chez-lee-Blanc, CH-1000 Lausanne 26, Switzerland ). The indicated temperature is the incubation temperature during the test. The medium indicated is the normal medium favorable to the growth of the strain to be tested.

TABEA VI TABLE VII TABLE VIII Species No. T (° C) Means Inhibition E. coli 1 30 BHI - Enterobacter chloacae 72 30 BHI - Salmonella ana tum XIV / 20 30 BHI - Salmonella typhimurium XIV / 274 30 BHI - Peeudomonae fluoreecens 3 30 BHI - The results illustrated in tables VI, vil and VII show the increased efficacy of the concentrate of the supernatant of the variacin culture, as an inhibitor of the growth of many strains that were tested in relation to what was supernatant. Inhibition spectra are observed for the same species and sub-species, but at a higher inhibition rate. This makes it possible to foresee the preparation of a concentrate of the supernatant of the variacin, as described in example 2, and its use for combating the pathogenic bacteria in the preparation of foodstuffs and cosmetic products, for example.

Resistance to DH: With the help of the Agar Well -Teet, it is determined if the variacin isolated according to the present invention is pH dependent. For this purpose, the agar is inoculated with an indicator strain, as described above, in the Well-Teet Agar test. It is used as an indicator strain: Lactobacilue helveticue (N 2). The pH of an extract of the concentrate of the supernatant of the culture described in Example 2 is adjusted, at a pH of 2 to 10 with 2N NaOH and / or 2N HCl, is incubated at 37 ° C for 60 minutes and then the pH is readjusted to 6-7 before depositing a sample of it in the Agar well. Teet. In parallel, a control is made, which is prepared with the same concentrate as the supernatant, at a pH of 7 and incubated at 37 ° C for 60 minutes, before depositing the control sample in the Agar Well -Teet well. way to compare the inhibition haloes with the control halo. The diameter of the inhibition halo of the control is 27 mm. In table IX, as in tables X and XI, the figure 27 indicates that there is still a halo of 27 mm corresponding to the full activity of the bactericidal power of the variacin.

PH Halos of inhibition 2 27 4 27 6 27 8 27 10 27 The results indicated in the table above, highlight the fact that the bactericidal power of variacin is not compromised. Therefore, it can be concluded that the bactericidal power of variacin is not pH dependent.

With the help of the iAgar Well -Teet, it is determined whether the variacin isolated according to the present invention is heat dependent. For this purpose, the agar is inoculated with an indicator strain, as described above, in the Well-Teet Agar test. It is used as an indicator strain: Lactobacilue helveticus (N 2). Incubate at 100 ° C for 15 to 60 minutes, a concentrate extract of the supernatant of the culture described in example 2, adjusted to a pH of 7 before depositing a sample thereof in the Well Agar well. Teet. In parallel, a control is made which is prepared with the same concentrate as supernatant at a pH of 7 and incubated at 37 ° C for 60 minutes. In this way, the control inhibition zones can be compared. The diameter of the inhibition halo of the control is 27 mm.

TABLE 2 Temperature (° C) Halting time of incubation inhibition (minutes) (mm) 100 15 27 100 30 27 100 60 27 These results show the fact that the variacin is not dependent on heat, end effect, the bactericidal power of the variacin is not compromised, even after an incubation at 100 ° C for 60 minutes.

Resistance to heat and to PH? In addition, the stability of the variacin is tested, combining the pH and the heat.

For this purpose, the agar is inoculated with an indicator strain, as described above, in the Well-Teet Agar test. It is used as an indicator strain: Lactobacilue helveticue (N 2). The concentrate extract of the supernatant of the culture described in Example 2 is adjusted to a pH of 4 to 7 with 2N HCl and / or NaOH, incubated at 115 ° C for 20 minutes and then readjusted again its pH at 6-7, before depositing a sample of it in the Well-Teet Well. At the same time, a control is carried out at a pH of 7 to 37 ° C for 20 minutes, before depositing the control sample in the Agar Well -Teet well, in order to compare the haloes of inhibition of the tests with the halo of inhibition of the control. The diameter of the inhibition halo of the control is 27 mm.

TABLE XI PH Temperature (° C) Halting time of incubation inhibition (minutes) (mm) 4 20 115 27 7 100 115 27 The results indicated in the table above show the fact that the bactericidal power of variacin is not compromised at a pH of 4 or 7, combined with an elevated temperature.

Purification of the variacin: 4 liters of BHI medium are inoculated with a culture of Micrococcue variane, producer of the variacin according to the present invention, this normal culture is incubated overnight at 30 ° C, under agitation in aerobiosis, and then centrifuged to 5000 g, in order to collect the supernatant in a container, in which 72 g of SAD-7 resin [AmJberlite (R)] are added. It is stirred at 25 ° C for 30 minutes, to facilitate the adhesion of the variacin molecules on the resin, and then the whole is transferred on a fritted glass where the supernatant is filtered under vacuum. The resin is washed successively in 3 buffers containing 20 mM sodium citrate pH 4, and isopropanol. The first buffer contains 10% isopropanol, the second buffer contains 15% isopropanol and the third buffer contains 20% isopropanol. The resin is transferred to a column and the variant is eluted with 700 ml of buffer containing 20 mM sodium citrate pH 4 and 25% isopropanol.

The bactericidal power of the variacin is controlled with the aid of the Agar Well -Teet as described in the foregoing. The active fractions are mixed and the isopropanol is evaporated. An S-Reeeource column for FPLC (Pharmacia) of 5 ml is prepared by equilibrating it with 20 mM sodium citrate buffer. The mixture of the active fractions, which was evaporated on this column, is charged, and then the content of the column is eluted with a NaCl buffer having a gradient of 100 M to 400 mM. The fractions are collected and the bactericidal power of the variacin purified in this way is verified with the help of the Agar Well-Teet.

Sequencing of the variacin: The N-terminal part of the purified variacin of strains CNCM 1-1586 and CNCM 1-1587 is sequenced, using an automatic sequencer 4774 of Applied Bioeystems. In this way, the peptide sequence of 5 amino acids whose sequence is identical to that for the N-terminal part of the sequence SEQ ID NO: 1 is disclosed, which is described in the list of sequences below.

It was not ible to reveal the presence of a peptide of more than 5 amino acids at the time of sequencing. In addition, a hydrolysis of the purified variacin of strains CNCM 1-1586 and CNCM 1-1587, with HCl for 10 minutes is carried out. 3 peptides are obtained which are classically isolated with HPLC. Only one of the three isolated peptides could be sequenced, because the other two certainly comprise peptide modifications. The sequence of that peptide that is thus isolated is identical to that comprising amino acids 19 to 22 of the sequence SEQ ID NO: 1, which is described in the list of sequences below. Finally, a fraction containing the purified variacin of the strains CNCM 1-1586 and CNCM 1-1587 is subjected to a mass spectrometry and a molecular weight for the variacin of 2659 Dal ton is revealed. mmMmmm m -____________ mAA * A homology between the sequence of lacticin 481 of Lactococcue lactie and that of variacin, according to the present invention was revealed. This homology is related mainly to the sequence of the N-terminal part of two bacteriocins. In fact, amino acids 1 to 5 of the N-terminal sequence of the variacin are identical to amino acids 3 to 7 of sequence SEQ ID NO: 8 of lacticin 481, which is described in the list of sequences below. However, it is only partial homology and not identity. In addition, a molecular weight of 2900 Dalton for excreted lacticin 481 is revealed, by means of mass spectrometry, while the molecular weight of the variant excreted is 2659 Dalton, as we have seen in the above. When the strain of Lactococcus lactie, producer of lacticin 481, is inoculated in the presence of an extract of the variacin, as it was already described in what proceeds in the inhibition spectrum test, an inhibition of the growth of that strain is observed . The strain of Lactococcue lactis, producer of lacticin 481, is immune to its own bacteriocin lacticin 481, but it is not against variacin, produced by one of the two strains of Micrococcue variane, according to the present invention. Those results that were obtained at the time of the inhibition test, which is described in the preceding, confirm the fact that it is not in the presence of two different bacteriocins. The genetic data that precede, show, the fact that lacticin 481 and variacin present sequence homologies, but are not identical. The biochemical data, as well as the icrobiological data, confirm the fact that lacticin 481 and variacin, are two different bacteriocins.

Sequencing of the variance sene: In a classical manner, the degenerate nucleotide sequence SEQ ID NO: 4 is manufactured which is described in the list of sequences below, which corresponds to the C-terminal part of the variant peptide sequenced in what precedes Then, the mixture of sequences SEQ ID NO: 4 becomes radioactive, through the action of T4 polynucleotide kinase. In a classical manner, a chromosomal DNA preparation of the strains CNCM 1-1586 and CNCM 1-1587 is carried out. A digestion of this DNA preparation is carried out by Salí, Sacl, Sphl, and BanHl, following the recommendations of the enzyme suppliers. Then, 2 μg of the digestion product is made to migrate on an agarose gel. The DNA of the gel is washed with 250 mM HCl and then the migration product is transferred in an alkaline medium of the gel onto a "Zetaproba" membrane (Biorad). Then, the Zetaproba membrane is prehybridized at a temperature of 55 ° C, lowered from 5 ° C every 2 hours to 40 ° C, in a medium comprising 6X SSC, 1% SDS, and 0.25% skim milk. This membrane is hybridized with the radioactive degenerate probe having the sequence SEQ ID NO: 4 in the hybridization medium which precedes and under the same temperature conditions. Then, it is allowed to incubate at 40 ° C for 4 hours, and then the membrane is washed at 40 ° C in 6X SSC. Finally, it is exposed to -80 ° C for 16 hours on an autoradiography film. Hybridization makes it possible to reveal different migration bands: a band of 7 kb Sal, a band of 1.4 kg Sacl, a band of 1.8 kb BamHl, and a band of more than 15 kb Sphl. Then, 5 μg of genomic DNA of strain CNCM 1-1586 is digested with the restriction enzyme BamHI and a 1.6-2 kb fragment is separated by chromatography on an agarose gel, followed by an elution of the part of the gel that binds it. contains The eluted DNA fragment is ligated to vector pK 19 [Gene, 56 (1987) 309-312] previously digested with BamHI and then treated with calf intestinal phosphatase (Boehringer Mannheim, part number 713023). Then, it is classically transformed with the binding medium, the strain Eecherichia coli BZ 234 (collection of Bíozentrum - University of Basel, Switzerland), which was made competent beforehand. The samples containing the insert are identified on an agar medium supplemented with 50 μm / ml of quanamycin, 60 mg / ml of IPTG (Boehringer Mannheim, part no 724815), 300 ng / ml of X-gal (Boehringer Mannheim, part no 651745 ) and incubated at 37 ° C for 16 hours. The white colonies that normally contain an insert are transplanted in microplates constituted by 96 wells. Each white colony is transplanted into one of the mentioned wells containing 150 μl of LB medium supplemented with 50 μg / ml of quanamycin and incubated at 37 ° C for 20 hours to produce mini-crops. Two starts of opposite orientation are prepared, due to the fact that the orientation of the gene in the pK 19 vector is not known. For this purpose, the aforementioned starts are constructed, making a montage from a nucleotide fragment having the sequence SEQ ID NO: 5 which partially codes for lacticin 481 which bind to each other of the universal probes of the pUC vectors which present the sequences SEQ ID NO: 6 or SEQ ID NO: 7. 1 μl of each well is mixed with 10 pmoles from one of the starts, 6 μl of dNTPs 2.5 mM μl buffer-Tag (PH Stehelin & amp;; Cie. AG, part TP05b), the whole is covered with a drop of Dyna-wax (Finnzimes Oy,, 02201 Espoo, Finland), and heated at 98 ° C for 10 minutes, in order to use the bacteria, and then the PCR. Then, it is lifted on an electrophoresis gel, a band of 800 bp for positive measurements. In this way, the positive samples are selected, the plasmid DNA is extracted from them and sequenced with the dideoxynucleotide method, the DNA fragment cloned in the vector pK 19 with the help of a sequencing ki t (Pharmacia Blotech , part no.27-1682-01) and of universal beginnings, and then of beginnings based on the sequence obtained in this way. In that way, a sequence SEQ ID NO: 2 is obtained which is described in the list of sequences below. That nucleotide sequence codes for the sequence SEQ ID N0: 1, which corresponds to the amino acid sequence of the variacin, before maturation. The examples below are presented by way of illustrations of the production process and the uses of the bacteriosine, according to the present invention. The percentages are given by weight unless otherwise indicated.

EXAMPLE 1: A BHI culture medium is inoculated to which a culture containing germs of the Micrococcue variane strain is added. The whole is incubated overnight at 30 ° C under agitation and put into aerobiosis after which, the medium contains 108 germs of the strain per me. The normal culture obtained in this way is centrifuged. What is normal is collected.

EXAMPLE 2: The supernatant concentrate obtained from 750 ml of the obtained supernatant is obtained, as described in example 1, to which 15 mg of XAD-7 resin [Amberli ta (R)] is added. The mixture is stirred for 60 minutes at 4 ° C and the mixture is then filtered through a Schleicher & Schuell (Germany) No. 604. The filter is then washed with 1% sodium citrate, in order to elute all the non-absorbed proteins, the resin is isolated, transferred in a vessel containing sodium citrate, the whole is stirred for 2 minutes. It is transferred with the sodium citrate in a column and the sodium citrate is eluted with 50% acetonitrile and 01% TFA. The eluate is evaporated and then resuspended in 50 mM phosphate buffer at a pH of 6.8.

EXAMPLE 3: 10 liters of a culture of the Micrococcue variane strain are produced in a BHI medium overnight at 30 ° C, under agitation and under aerobic conditions. 200 g of the XAD-7 resin (Amberli te) are then added directly to the culture, and the whole is stirred slowly for 1 hour at 4 ° C. Then, the mixture is filtered through a Schleicher & Schuell (Germany) No. 604, and then the resin that was retained on the filter was washed with 10 liters of a 50 mM acetic acid solution pH 5.2, in order to remove the bacteria. Then, 450 ml of a solution comprising 100% ethanol and 20 mM ammonium acetate are added to the resin, the whole is filtered to remove the resin, and then the filtrate is lyophilized to a powder comprising the variacin according to the invention. invention, which can be used in the food industry. Then, the bactericidal power of this powder previously diluted in water is determined, with that of the agar test well described above. This powder has at least 105 uua / g of powder. Finally, 0.5 g / kg of the powder mentioned above is added to a meat foam, in the course of its preparation in the traditional manner. Therefore, a meat foam is obtained comprising 50 ua / g of bacteriocins that can totally inhibit the development of pathogenic bacteria, especially Cloetridia.

EXAMPLE jx This example relates to the preparation of a skin care moisturizer, which contains 0.05 g / kg of the powder described in example 3, or is therefore variacin that can inhibit the development of undesirable bacteria on the skin. the skin, in particular Staphylococcus aureue and Streptococcus pyogenee, at a rate of 5 ua / g. To manufacture this emulsion, the components of the lipid phase A are mixed and heated to 75 ° C. The aqueous phase B is prepared, and it is also heated to 75 ° C, and then it is added to the liquid phase with slow stirring, and then the whole is cooled under slow stirring to room temperature, at about 25 ° C. At this temperature, the constituents C are slowly added in the order of the formula.

Lipid phase A: Peg-6-stearate, glycerate and peg-20-cetyl ether 15 (peg: polyethylene glycol) Vaseline oil 5 Stabilized wheat germ oil with 0.1% phenylindane 3 (antioxidant) and 1% soybean phospholipids (see EP-94.109355.1) Sweet almond oil 2 Cetyl alcohol 1 Isostearyl isostearate 2 2-Octyl-docecyl-mistirate 1 Lanolin wax 1 Aqueous stream B: Methylisothiazoline 0.1 Demineralized water 59.6 Human placenta protein Additive C: Propylene glycol and calendula extract 2 50% collagen soluble in demineralized water 5.8 Perfume 0.3 2. 5% bacteriocin powder according to example 3 in demineralised water 0.2 EXAMPLE 5 0.5 g / kg of the bacteriocins powder described in example 3 is added to a toothpaste. This way, this tooth water can inhibit the development of pathogenic bacteria of the oral cavity, and especially Strectococcus sobrinus, streptococcus sanguis, Streptococcus mutans and Actinomycee viscosue.

EXAMPLE 6 A solution comprising the bacteriocin powder of example 3 diluted in water at a rate of 1% on a food product intended to be sterilized is sprayed to prevent post-contamination at the time of packing. (1) General Reports: (i) Applicant; (A) Name: SOCIETE DES PRODUITS NESTLE (b) Street: Avenue Nestlé 55 (C) City: VEVEY (D) State or Province: Canton of VAUD (E) Country: SWITZERLAND (F) Postal Code: 1800 (G) Telephone : (041) 21 924 34 20 (H) Telecopy: (041) 21 924 28 80 (ii) Title of the invention: BACTERIOCINA DE MICROCOCCUS VARIANS. (iii) Number of sequences: 8 (iv) Computer decodable form: (A) Type of support: Floppy disk (B) Computer: IEM compatible PC (C) Operating system: PC-DOS / MS-DOS (D) Program: Patent in Releaee # 1.0, Version # 1.30 (OEB) (2) Reports for SEQ ID NO: 1: (i) Sequence characteristics: (A) Length: 25 amino acids (B) Type: amino acid (C) Number of strands: (D) Configuration: linear (ii) Type of molecule: protein (vi) Origin: (A) organism: MICROCOCCUS VARIANS (C) INDIVIDUAL / ISOLATED: Dos CNCOS 1-1586 and CNCM 1-1587 (xi) Description of the sequence: SEQ ID NO: 1: Gly be Gly Val He Pro Thr I have to be His Glu Cys His 1 5 10 Met Asn Ser Phe Gln Phe Val Phe Thr Cye Cys Ser 15 20 25 (2) Reports for SEQ ID NO: 2: (i) Sequence characteristics: (A) Length: 278 base pairs (B) Type: nucleotide (C) Number of ends: double (D) Configuration: linear ( ii) Molecular type: DNA (genomic) (vi) Origin: (A) Organism: MICROCOCCUS VARIANS (C) INDIVIDUAL / ISOLATED: Doses CNCM 1-1586 and CNCM 1-1587 (ix) Feature: (A) Name / key: CDS (B) Location: 88.228 (ix) Characteristic: (A) Name / password: sig-peptide (B) Location: 88..153 (ix) Characteristic: (A) Name / password: mat- peptide (B) Location: 154..228 (xi) Sequence description: SEQ ID NO: 2: GTTGAAAATC ATCCGGAAGG ATGATTCTGT GTTCATGCGA GGCCACCCGG AATCGCGGCA 60 GCCACACCCA CTGAAGAGGAC TAGAACA ATG ACG AAC Met Thr Asn -22 -20 GCA TTT CAG GCA CTG 111 Ala Phe Gln Ala Leu -15 GAC GAA GTC ACG GAC GCC GAG CTC GAC GCC ATC Asp Glu Val Thr Asp Ala Glu Leu Asp Ala He -10 -5 CTT GGC GGG GGC AGT 159 Leu Gly Gly Gly be 1 CGT GTT ATT CCC ACG ATC AGC CAC GAG TGC CAC Gly Val He Pro Thr He Ser His Glu Cye His 5 10 ATG AAC TCC TTC CAG 207 Met Asn Ser Phe Gln 15 TTC GTG TTC ACC TGC TGC TCC TAGAGAAACTC Phe Val Phe Thr Cys Cys Ser 20 25 CTCCGATGCT CAGAGGGCCG 258 CGCTAGGAAA ATCTAGTAAG 278 (2) Reports for SEQ ID NO: 3: (i) Sequence characteristics: (A) Length: 47 amino acids (B) Type: amino acid (D) Configuration: linear (ii) Type of molecule: protein (xi) Sequence description: SEQ ID NO: 3: Met Thr Asn Ala Phe Gln Ala Leu Asp Glu val Thr Asp Ala -22 -20 -15 -10 Glu Leu Asp Ala He Leu Gly Gly Gly Ser Gly val He Pro -5 1 5 Thr He Ser His Glu cys His Met Asn Ser Phe Gln Phe Val 10 15 30 Phe Thr Cys cys Ser 25 (2) Reports for SEQ ID NO: 2: (i) sequence characteristics: (A) Length: 23 pairs of bases (B) Type: nucleotide (C) Number of ends: simple (D) Configuration: linear (ii) Type of molecule: Other nucleic acid (A) Description: / desc = "oligonucleotide" (iii) Hypothetical: Yes ( xi) Description of the sequence: SEQ ID NO: 4: TGGCARTTYG TNTTYACNTG YTG 23 (2) Reports for SEQ ID NO: 5: (i) Sequence characteristics: (A) Length: 44 base pairs (B) Type : nucleotide (C) Number of ends: simple (D) Configuration: linear (ii) Type of molecule: Other nucleic acid (A) Description: / desc = "oligonucleotide" (xi) Description of the sequence: SEQ ID NO: 5 : GGNTCNNGGNG TNATHCCAYAC NATHTCNCAY GARTGYAAYA TGAA 44 (2) Reports for SEQ ID NO: 6: (i) Sequence characteristics: (A) Length: 18 base pairs (B) Type: nucleotide (C) Number of ends: simple (D) Configuration: linear ( ii) Type of molecule: Other nucleic acid (A) Description: / desc = "oligonucleotide" (xi) Description of sequence: SEQ ID NO: 6: ATAACAATTT CACACAGG 18 (2) Reports for SEQ ID NO: 7: ( i) Sequence characteristics: (A) Length: 18 base pairs (B) Type: nucleotide (C) Number of ends: simple (D) Configuration: linear (ii) Type of molecule: Other nucleic acid (A) Description : / desc = "oligonucleotide" (xi) Description of the sequence: SEQ ID NO: 7: GGTTTTTCCCA GTCACGAC 18 (2) Reports for SEQ ID NO: 8: (i) Sequence characteristics: (A) Length: 7 amino acids (B) Type: amino acid (C) Number of ends: simple (D) configuration: linear (ii) Molecular type: Peptide (xi) Sequence description: SBQ ID NO: 8 Lys Gly Gly Ser Gly Val He 1 5

Claims (11)

1. NOVELTY OF THE INVENTION Having described the invention, we consider it as a novelty and, therefore, we claim as of our novelty, what is contained in the following clauses. 1. Bacteriocin of Micrococcue variane characterized in that it presents the amino acid sequence SEQ ID NO: l.
2. 2. Bacteriocin according to clause 1, characterized in that it differs from the sequence SEQ ID N0: 1 by a substitution, a deletion and / or an insertion of 1 to 4 amino acids.
3. 3.- Coding nucleotide fragment for the bacteriocin of Micrococcue variane according to any of clauses 1 and 2.
4. 4.- Fragment according to clause 3, characterized in that it comprises nucleotides 88 to 228 of the sequence SEQ ID NO: 2.
5. 5. - Fragment according to clause 3 or 4, characterized in that it comprises nucleotides 154 to 228 of the sequence SEQ ID NO: 2.
6. 6.- Fragment according to clause 3 or 4, characterized in that it comprises nucleotides 88 to 153 of the sequence SEQ ID NO: 2.
7. 7.- Micrococcue variane signal peptide encoded by the nucleotide sequence according to clause 6.
8. 8. - Micrococcue varians strain producing the bacteriocin according to clause 1 or 2, especially the strains CNCM 1-1586 and CNCM 1-1587.
9. 9.- Procedure for preparing bacteriocin according to clause 1 or 2, characterized in that a strain of Micrococcue variane producing this bacteriocin is cultivated in medium and in favorable growth conditions, in order to obtain a culture medium containing 107 1011 germs of that strain for me, the culture that was obtained is centrifuged, and then an extract of what supernatant that bacteriocin contains is prepared.
10. 10. Procedure according to clause 9, characterized in that the strain of Micrococcue variane is the strain CNCM 1-1586 or the strain CNCM 1-1587.
11. 11.- Use of bacteriocin according to clauses 1 and 2, especially in the form of extract obtained according to clause 9 and / or of a strain of Micrococcue varians that produces that bacteriocin, in the preparation of food products, of products cosmetics and pharmaceutical products, in particular in dermatology and in dental-dental applications. The invention relates to a bacteriocin from Micrococcus varians, a strain producing this bacteriocin, especially strains CNCM 1-1586 and CNCM 1-1587, a method for preparing this bacteriocin and the use of this bacteriocin as an active agent against pathogens in the preparation of food products and cosmetic products.