The bacteriocins, method thereof, a strain of micrococcus varians, a nucleotide fragment, signal peptide

 

(57) Abstract:

The invention relates to biotechnology and concerns bacteriocin from Micrococcus varians, a nucleotide fragment encoding the bacteriocins, strains of Micrococcus varians CNCM 1-1586 and CNCM 1-1587 producing the specified bacteriocins, as well as method of obtaining bacteriocin and its use to obtain food and cosmetics as an agent, active against pathogens. The bacteriocins of the present invention has a strong bactericidal effect, and has a wider range of inhibition of different bacterial strains. 6 C. and 8 C.p. f-crystals, 11 PL.

The invention relates to bacteriocins, to the strain that produces the mentioned bacteriocins, to a method for bacteriocin and to the use of such bacteriocin and/or strain producing the above-mentioned bacteriocins in food and cosmetics.

Bacteriocins isolated from numerous gram-positive and gram-negative bacteria. Bacteriocins are molecules, by their nature, reminiscent of the protein, which have a bactericidal effect, and for this reason, the bacteriocins causes antagonistic reaction between bacteria, it produces, and DAMI, similar to the strain that it produces.

The bacteriocins were, in particular, found in lactic acid bacteria. For example, in patent EP 0643136 (Societe des produits Nestle) describes the identification of two bacteriocins from Streptococcus thermophilus. Similarly was selected bacteriocins from Lactobacillus lactis (Arr. and Env. Environ., 1992, 58, 279-284; J. of Biol. Chem., 1993, 268, 16361-16368).

However, to date, not known bacteriocins derived from Micrococcus varians. Micrococcus varians currently widely used in food production, in particular during the fermentation of meat with the aim of obtaining a delicatessen products, such as salami and other sausages. In this regard, it is important to have available producing bacteriocins strain of this bacteria in order to fight against pathogens.

The present invention is directed to solving this problem.

Described in the present invention, the bacteriocins represents the bacteriocins from Micrococcus varians, these bacteriocins represents the amino acid sequence of SEQ ID No: 1 or any amino acid sequence that differs from the sequence SEQ ID No: 1 by one substitution, one deletion and/or insertion of from 1 to 4 amino acids.

Described in the present invention, the strain also applies to the strain of Micrococcus varians, which produces the specified bacteriocins, in particular to the strains CNCM I - 1586 and CNCM I-1587 Micrococcus varians.

By way of obtaining bacteriocin in accordance with the present invention a strain of Micrococcus varians, which produces bacteriocins, in particular, the strain CNCM I-1586 or strain CNCM I-1587, cultured in a suitable growth of the microorganism environment compliance with favourable conditions, allowing to obtain a culture medium containing from 107up to 1011organisms need strain per ml, after which the culture medium centrifuged, and the extract supernatant containing bacteriocins, are used for the further steps of receiving the drug. Finally, the present invention describes the use of bacteriocin from Micrococcus varians, which includes the use of its nucleotide sequence, as well as signal sequences, extract supernatant containing bacteriocins, as well as strain Micrococcus varians, which produces bacteriocins in food, cosmetics and pharmaceutical about the research Institute of the invention bacteriocins designated by the term "variation".

In the framework of the present invention the conventional unit (UE) is defined as the reciprocal of the highest dilution at which the sample still exhibits a bactericidal effect in the test, which every expert in the field known as "agar test.

In the framework of the present invention, the term "fragment" or "fragment of DNA" should be understood as a fragment of single-stranded or double-stranded DNA that is partially or completely coded and which can be synthesized, replicated in vitro using, for example, a known method of polymerase-cableway reaction, or replicated in vivo in bacteria, such as Escherichia coli.

In the framework of the present invention, the term "homologous fragment" should be understood as any fragment that differs from the fragments of the present invention semimedium, deletion or insertion of a small number of bases. In the context of the present invention two fragments of DNA that encode the same polypeptide, due to the degeneration of the genetic code will be considered as homologous. As a homologous fragment is also considered a snippet that shows more than 80% homology with a fragment of this isopreference and their number in the fragment of the present invention.

And, finally, in the framework of the present invention, the term "fragment's hybrid" should be understood as any fragment that is capable of hybridization with fragments of the present invention according to the method of southern Blotting (Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, U. S. A., 1989, chapter to 9.31 9.58). Preferably the hybridization is performed under conditions sufficiently stringent, in order to avoid non-specific hybridization or hybridization, which is relatively unstable.

Proteinopathy factor, in this case, the bacteriocins with a strong bactericidal effect, was isolated from the strains CNCM I-1586 and CNCM I-1587. The bacteriocins, which was isolated from Micrococcus varians and which, therefore, presents the amino acid sequence SEQ ID No: 1 shown in the sequence listing below, was named variation.

In connection with the properties of Mariazinha, the present invention relates to any bacteriocins, which has an amino acid sequence that differs from SEQ ID No: 1 by one substitution, one deletion and/or insertion of from 1 to 4 amino acids. Thus, the above mentioned bacteriocins, which has an amino acid sequence that differs is t to have a wider range of inhibition for bacterial genus or bacterial strains, than, for example, mentioned variations.

It is also possible to carry out the selection of chromosomal nucleotide fragment encoding variations of the present invention, of the two strains CNCM I-1586 and CNCM I-1587. Both of the mentioned fragment describes the sequence of SEQ ID No: 2, shown below in the list of sequences.

The interest of the present invention is any nucleotide fragment encoding variation of the present invention, in particular, nucleotide fragments which are homologous to the sequence SEQ ID No: 2 or hybridize with it.

In particular, the present invention relates to a nucleotide with 88 153 in the sequence SEQ ID No: 2, which encode the signal peptide Mariazinha, to the nucleotides from 154 to 228 in the sequence SEQ ID No: 2, encoding the secretory variation of the present invention, and/or nucleotides with 88 228 in the sequence SEQ ID No: 2, encoding the bacteriocins, fused with a signal peptide.

The fragment encoding the secretory variation, can be used for the expression of Mariazinha of the present invention in different organisms, different from Micrococcus varians. In this case, the nucleotides from 154 to 228 in the sequence and to the left of the terminator, given the reading frame, and then the above vector can be introduced into a plant, bacteria or yeast in order to increase the range of inhibition include, for example, certain bacteria.

You can make use of the signal sequence of the present invention through the merger of the plot, with 88 153 nucleotides in the sequence SEQ ID No: 2 with a gene of interest, given the reading frame, and then clone it completely into the expression vector Micrococcus varians to allow the protein encoded mentioned interesting gene to be expressed and to secretariats, for example, Micrococcus varians.

Nucleotides with 88 228 in the sequence SEQ ID No: 2 can be cloned in the expression vector Micrococcus varians and put then in another strain of Micrococcus varians, so the last specified strain begins to produce variation of the present invention.

In addition, a strain of Micrococcus varians, which contains either integrated into its genome, or through the participation of expression vector DNA fragment encoding variations of the present invention, is a part of the present invention. In particular, strains of Micrococcus varians, deposited June 7, 1995, in accordance with the Budapest Treaty at the National the Pasteur, 25 Rue du Docteur Roux, F-75724 PARIS CEDEX 15, France), where they received the Deposit number CNCM I-1586 and CNCM I-1587, are part of the present invention.

Bacteria Micrococcus varians are gram-positive and positive for catalase aerobic bacteria, which is permanently immobilized. They have a spherical shape and are found in the form of tetrad randomly grouped.

Colonies of Micrococcus varians on WN environment (with cardio-cerebral hood painted in yellow color. The optimum temperature for growth of these strains is 25-37oC.

The strains CNCM I-1586 and CNCM I-1587 covered by the present invention, metabolize and glucose, and fructose. And the strain CNCM 1-1587 also metabolizes sucrose and turanose.

In addition, the strain CNCM I-1586 carries two plasmids with sizes of 4 and 12, etc., ad , whereas the strain CNCM I-1587 has a single plasmid size 7, etc., N.

Cultural supernatant strains CNCM I-1586 and CNCM I-1587 relatively wide range of inhibiting the growth of other bacteria, among which we can mention, for example, the following: Lactococcus lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulg-aricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus sake,vegetative cells of Bacillus subtilis, Bac. illus cereus. Bacillus polyinyxa. Bacillus circulans. Bacillus pumulus and Bacillus liqueniformis and Clostridia.

Preferably, in the method of producing Mariazinha, strain Micrococcus varians, producing variation, cultivated in an environment and under conditions favorable for its growth, in order to obtain a culture medium containing from 107up to 1010organisms mentioned strain per ml, after which the resulting culture is centrifuged, and the extract supernatant containing the described bacteriocins, is subjected to further processing.

To obtain the extract of the strain Micrococcus varians of the present invention, producing variation, in particular the strain CNCM I - 1586 or strain CNCM I-1587, can be cultivated in the environment and compliance with those conditions that are favorable for the growth of Micrococcus varians. In this case, the cultivation may be carried out in BHI medium at a temperature 25-37oC in aerobic conditions on the rocker until the desired concentration of organisms, for example, from 107up to 1011per milliliter of the medium. Derived standard the culture is then centrifuged at 4000 - 6000 g, and the extract supernatant containing the described bacteriocins, collect.

The present invention relates also to the use of Mariazinha, is ozin, for the production of food and cosmetic products.

The culture of one of the mentioned strains of Micrococcus varians of the present invention can be, in particular, used in the fermentation of meat for cooking salami, as well as to fight infection, such as Clostridium.

Variation in the form of crude extracts or in purified form can be used when adding to the ferment, which is obtained by the use of bacteria resistant to the mentioned variatio, in the process of making cheese, such as cheese like mozzarella, in order to avoid the cheese holes produced by Bacillus poymyxa spores which survive in the fermentation process, and the type vacherin in order to combat the pollution caused, for example, Listeria monocytogenes.

Variation in crude extracts or in purified form, or one of the two producing its strains can be used as an additive or agent, active against pathogenic bacteria, to get dessert mousses, such as pasteurized creams, in order to prevent growth of spores of microorganisms such as Clostridia and Bacillus cereus, or growth of these bacterial strains as Listeria.

In addition, variation in rough extrac is, is active against pathogenic bacteria, for the production of cosmetic products such as moisturizing creams or deodorants in order to fight pathogenic bacteria, pollutants, in this case the skin.

Variation of the present invention described in more detail below with the help of various microbiological, biochemical and genetic data that illustrate its properties. The above percentages are weight percent.

Unit bacterial activity - "agar test.

In the context of the present invention bactericidal activity is defined in terms of conventional units.

The supernatant standard culture of Micrococcus varlans of the present invention, prepared under the conditions described for example, in example 1, has in a typical case, the activity 640 UE/ml Similarly, the concentrate of the above supernatant, which is obtained under the conditions described, for example, in example 2, is usually a activity 24000 UE/ml

Mentioned agar test used to determine whether the sample at a given value of dilution bactericidal activity.

To conduct this test, 15 ml of MRS agar medium containing the indicator strain in to what you learn Lactobacillus bulgaricus (YL 5) or Lactobacillus helveticus (N2) is used as the indicator strain.

In culture medium make holes with a diameter of 5 mm test specimens supernatant or concentrated supernatant poured into these holes 50 Midi on the hole. Cup put on a pre-incubation at 4oC for 2 hours and then incubated overnight at a temperature of 30oC or 37oC, depending on the type of indicator strain. After the growth of the indicator strain become visible halos of inhibition. The magnitude of the dilution at which the sample no longer shows bactericidal activity represents the value of the dilution from which the halo of inhibition is no longer visible.

Inactivation of enzymes

Mentioned agar test is used to determine inactivated whether variation allocated in accordance with the present invention, in the presence of a proteolytic enzyme or in the presence of catalase.

In order to conduct this test, with a speed of 5 mg/ml to concentrate the culture supernatant as described in example 2, add the enzyme. Create conditions for the validity of the EN zymes is correctly prepare the control using the same concentrate at pH 7 and without added enzyme. Control sample incubated at temperature 37oC for 60 minutes before placing it in the agar plate test in order to compare halo of inhibition obtained in the presence of the enzyme, with a halo of inhibition in control. Diameter control of a halo of inhibition is 27 mm

In table. 1 shows the results obtained with enzymes, which are investigated with the use of indicator strain Lactobacillus helveticus (N2). In this table, as in table. II, the enzyme is indicated by its type, supplier name and number in the vendor catalog. The number Of points to the absence of a halo of inhibition, in other words that the bactericidal effect of bacteriocin suppressed by incubation with the enzyme. Number 27 indicates that there is a halo of inhibition of size 27 mm, corresponding to the bactericidal activity of Mariazinha.

In table. II shows the results that were obtained for the enzymes investigated using the indicator strain Lactobacillus bulgaricus (YL 5).

The results are shown in table. I and II show that all proteolytic enzymes inhibit the bactericidal activity of Mariazinha. They suggest that variation for their bactericidal activity.

The fact that, according to the results of studies of the effects of catalase on the bactericidal activity of Mariazinha indicates that the inhibition of growth of two indicator strains is not due to the antibacterial activity of H2O2similar to the activity of bacteriocins, since H2O2would be subject to degradation by catalase.

The range of inhibition of the culture supernatant containing variation

Was applied agar test in order to determine whether the antibacterial activity of the culture supernatant containing variation of the present invention, the inhibitory activity against the growth of various strains of spores and bacteria. Thus was defined range inhibition supernatant.

To conduct these tests in 15 ml of standard medium containing 15 μl of the supernatant of the studied culture, which was obtained during the night of cultivation before, put on Petri dishes so as to obtain a bacterial concentration in the range of 105-1061 ml of standard medium. Under standard environment refers to an environment which is favorable for the growth of the studied strain. If the investigated sredy.

In each Petri dish make holes with a diameter of 5 mm According to the method described in example 1, each of them contribute samples of culture supernatant in 50 ál. Cup put on a pre-incubation at a temperature of 4oC for 2 hours and then incubated at a temperature conducive to the growth of the investigated strain in the test conditions during the time needed to cover a Cup of visible bacterial lawn.

The effect or the degree of inhibition is measured by the diameter of the observed halo of inhibition. Inhibition is assessed as very strong (++++), if the halo has a diameter of 18-28 mm, strong ( + + + ) if the halo has a diameter of 10 to 17 mm), medium (++), if the diameter of 5-9 mm, weak (+), if the diameter of 1-4 mm and a null ( - ) if the halo of inhibition is not detected.

By the above method were investigated 32 strains of different species and subspecies of lactic acid bacteria, and none of them showed resistance to the supernatant. In table. III provides more detailed results of the study. In it, as in subsequent tables, the designation of strain or numbers refer to the numbers assigned to the strain in the Collection microorganizmelor mean incubation temperature within the test. And this environment means a standard environment conducive to the growth of the studied strain.

From the data table. III shows that the range of inhibition of the supernatant is quite wide, because the level of inhibition has approximately the same degree of severity in different studied species.

The range of inhibition of the culture supernatant, producing variations of the present invention is also seen as broad as it is not limited to lactic acid bacteria, and includes, as is seen in table. IV results, other gram-positive bacteria, in particular, undesirable or pathogenic bacteria, such as Listeria innocua, Listeria welhia, Listeria monocytogenes and spores, for example. Bacilli, as can be seen from the table. IV.

Are given in table. The IV results suggest the possibility of using the described supernatant or purified Mariazinha as an additive in the manufacture of food products, which will act as agent, active against pathogenic bacteria, in particular against Clostridia, in meat products, against Listeria monocytogenes in cheeses, against Bacillus cereus and Listeria in dessert mussah, or against Bacilli in the fresh pastes or sauces for Soldatov, are given in table. V, the variation does not exert any inhibitory effect on the growth of gram-negative bacteria.

Range inhibition concentrate supernatant containing variation

Procedure coincides with the above-described method, except that determine the inhibiting effect of concentrate of the supernatant obtained by the method of example 2, on the growth of different strains of spores and bacteria.

Explore the same species and subspecies, as before. The results of the tests are shown in table. VI, VII and VIII. The specified strain designation or number matches the number assigned to the strain in the Collection of microorganisms Nestle, Switzerland (Nestle, Address: NESTEC S.A. Centre de Recherche, Verz-chez-les-Blanc, CH-lOOO Lausanne 26, Switzerland). This temperature refers to the temperature of incubation, supported during testing. Specified in tables environment refers to the standard environment conducive to the growth of the studied strain.

The results are shown in table. VI, VII and VIII indicate improved performance of the concentrate of the supernatant compared with the supernatant in the inhibition of growth in many of the investigated strains. The spectrum of inhibition observed for the same species and subspecies, but the nature of the terrain obtain, according to the method of example 2, the concentrate of the supernatant containing variation, and its application to fight against pathogenic bacteria in the preparation of, for example, food products and cosmetics.

Resistance to pH

To determine whether variation allocated according to the method of the present invention, pH-dependent, is also used agar test.

In this case, the agar cause the indicator strain in compliance with all conditions outlined in section agar test. As the indicator strain used Lactobacillus helveticus (N2).

the pH of the extract concentrate the culture supernatant as described in example 2 was adjusted to a value of from 2 to 10 with the help of 2n NaOH and/or 2n HCL, extract incubated at temperature 37oC for 60 minutes, after which the pH again adjusted to 6-7, and then in agar well make the sample extract to conduct agar test.

In parallel survey control using the same concentrate of the supernatant at pH 7, which is incubated at a temperature of 37oC for 60 minutes and then control sample contribute in agar well in order to compare the zones of inhibition of test samples and control. Zone diameter podklinivanija size 27 mm, that shows a complete bactericidal activity of Mariazinha.

Are given in table. IX the results show that the bactericidal activity of bacteriocin not suppressed. Therefore it can be concluded that the bactericidal activity of Mariazinha does not depend on pH.

The heat resistance of

To determine whether variation allocated according to the method of the present invention, heat-dependent, is also used agar test.

In this case, grown on agar indicator strain, compliance with all conditions outlined in section agar test. As the indicator strain used Lactobacillus helveticus (N2).

The extract concentrate the culture supernatant as described in example 2, brought to pH 7, and incubated at a temperature of 100oC during the period of time from 15 to 60 minutes, after which the pH was adjusted to 6-7 and then in agar well make the sample extract to conduct agar test.

In parallel survey control using the same concentrate of the supernatant at pH 7, which also incubated at temperature 37oC for 60 minutes and then in agar well make a control sample. Provideany inhibition control is 27 mm

Are given in table. X data indicate that the activity of Mariazinha does not depend on temperature. Thus, the bactericidal activity of Mariazinha not suppressed even after 60-minute incubation at a temperature of 100oC.

The stability of Mariazinha heat is a very important biochemical feature, which is set for use in obtaining food and cosmetics. Thus, variations can be used as a rough extracts or in purified form when receiving, for example, pasteurized food products, in order to protect them from the germination of spores, in particular Bacilli, which are thermotolerance.

Stability to heat and pH

Activity Mariazinha examine, in addition, the combination of two factors: pH and temperature.

In this case, grown on agar indicator strain, compliance with all conditions outlined in section agar test. As the indicator strain used Lactobacillus helveticus (N2).

The extract concentrate the culture supernatant as described in example 2, brought to pH 4 or 7 with 2n HCl and/or 2n NaOH, incubated at a temperature of 115oC for 20 minutes, after which Esta.

Parallel conduct a study of the control of pH 7, which is incubated at a temperature of 37oC for 20 minutes and then in agar well make a control sample in order to compare the zones of inhibition of test samples with the control zone. The diameter of zone of inhibition control is 27 mm

Are given in table. XI the results show that the bactericidal activity of Mariazinha not inhibited at pH 4 or 7 in the case of a combination of the pH-value with increasing temperature.

Cleaning Mariazinha

In 4 l of BHI medium inoculant culture of Micrococcus varians, which produces variation of the present invention. The specified standard culture is incubated at a temperature of 30oC during the night in aerobic conditions during the swing, after which it was centrifuged at 5000 g and the supernatant is collected in the receiving vessel, to which is added 72 g of resin XAD-7 (Amberlite (R)).

The mixture is stirred at a temperature of 25oC for 30 minutes to facilitate adhesion molecules Mariazinha with resin and then all together transferred into a vessel made of sintered glass, and then under vacuum filtered supernatant.

The resin is successively washed with 3 buffers containing 20 mm sodium citrate, pH 4 iufer contains 20% isopropanol.

The resin is transferred onto the column, followed by the elution of Mariazinha with 700 ml of buffer containing 20 mm sodium citrate, pH 4 and 25% isopropanol. Constantly monitor bactericidal activity using the above agar test.

Mix the active fractions and evaporate the isopropanol. Prepare 5 ml S-Resource S-Resourse) column for HPLC [Pharmacia (Pharmacia)], balancing its 20 mm sodium citrate buffer. One stripped off the mixture of the active fractions is applied to the column, followed by the elution of the content column NaCI-buffer in a gradient from 100 mm to 400 mm.

Collect fractions and using agar test check bactericidal activity thus purified of Mariazinha.

Sequencing of Mariazinha N-terminal segment of Mariazinha isolated from strains CNCM I-1586 and CNCM I-1587 and then purified, is sequenced using automated sequencing machine Applid of Biosystems 4774 (Applied Biosystems 4774).

The analysis revealed that the peptide sequence of 5 amino acids identical to the N-end sequence of SEQID No: 1 below in the sequence listing.

It was not possible during sequanian is selected from the strains CNCM I-1586 and CNCM I-1587 and subsequently purified, hydrolyzing under the action of 6N HCl for 10 minutes. There were obtained 3 peptides that are analyzed in the usual way using HPLC. It was possible to carry out sequencing of only one of the three selected peptides, as the other two, apparently, include modification of peptides.

The sequence underlined peptide was identical to the plot, including amino acids 19 to 22 in the sequence SEQ ID No: 1, shown below in the list of sequences.

Finally, the fraction containing variation, isolated and subsequently purified from the strains CNCM I-1586 and CNCM I-1587, explore a method of mass spectrometry, the result of which was determined molecular weight of Mariazinha equal 2659 Dalton.

Homology

Between sequences of lacticin 481 from Lactococcus lactis and Mariazinha of the present invention was observed homology. Homology is concerned, in particular, sequences of N-terminal sections of both bacteriocins. Thus, the sequence from 1 to 5 amino acids at the N-terminal segment of Mariazinha identical to the area from 3 to 7 amino acids in the sequence SEQ ID No: 8 lacticin 481, which will be described below in the sequence listing. is.

In addition, as was shown by mass Spectro-metric analysis, the indirect lacticin has a molecular weight equal to 2900 daltons, whereas the molecular weight of secreted Mariazinha, as mentioned, is 2659 Dalton.

In the case of insulinopenia according to the method previously described in the test for determining the spectrum of inhibition, in the presence of extract Mariazinha, strain of Lactococcus lactis producing lacticin 481, shows that the growth of the above-mentioned strain inhibited. The strain of Lactococcus lactis producing lacticin 481, immune to his own bacteriocins-lacticin 481, however, he is not immune to variatio produced in one of two strains of Micrococcus varians of the present invention. Thus, the results obtained using the above test on the determination of the spectrum of inhibition, confirming the idea that both of these bacteriocin different.

The above genetic data show that, despite the fact that lacticin 481 and variations show homology sequences, their sequences are not identical. Biochemical and microbiological data support the idea that lacticin 481 and variations javlautsa sequence of SEQ ID No: 4, which is described below in the sequence listing, and which corresponds to the C-terminal portion of peptide sequenced previously variazione, designed in a known manner. The mixture is then sequences SEQ ID No: 4 radioactively irradiated with the participation of polynucleotide kinase from T4.

The traditional way get a drug chromosomal DNA of the strains CNCM I-1586 and CNCM I-1587. The above-mentioned DNA preparation is subjected to digestion by enzymes SalI, SacI, Sphi and BamHI in accordance with the recommendations given in the instructions of the supplier. After that, the agarose gel is to be applied 2 µg product of digestion. In the gel the DNA was washed with 250 mm HCl, after which the product movement is transferred into the alkaline environment of the gel on the membrane of Thetaprobe" (BioRad) ["Zetaprobe" (Biorad)]. After that, membranes "Thetaprobe" subjected to pre-hybridization at a temperature of approximately 55oC subsequent reduction of the indicated temperature for 5oC every 2 hours to a temperature of 40oC in a medium containing 6 x SSC solution chloride and sodium citrate, 1% LTOs (SDS) and 0.25% skim milk. The specified membrane further hybridizing with degenerative radioactive breakdown, representing a sequence of SEQ ID No: 4 in the previously described hybridization environment within 4 hours, then the membrane is washed at a temperature of 40oC in 6 x SSC. Finally, it is applied to the film for autoradiography at - 80oC for 16 hours.

The hybridization results demonstrate the presence of multiple bands of migration: SalI band size 7, etc., ad, SacI stripe size of 1.4 T. p. N., BamHI band 1.8 T. p. N. and SphI band larger than 15 tons, p. N.

Then, 5 μg of genomic DNA of strain CNCM I-1586 digested with restriction enzymes BamHI and separated fragment of 1.6-2, etc., N. chromatography in the agarose gel, followed by elution of the portion of the gel, which includes the desired fragment. Suirvey DNA fragment are ligated with the vector RK (Gene, 56 (1987) 309 to 312), which was previously digested BamHI and then treated with phosphatase from calf intestine [Boehringer Mannheim (Boehringer Mannheim), part N 713032].

Then the traditional way spend the transformation of Escherichia coli, strain BZ 234 [Collection Biozentrum, (Blocentrum collection), University of Basel, Switzerland] , which is the competent environment for ligating. Clones containing the insert, identify agar medium containing 50 μg/ml kanamycin, 60 ng/ml IPTG (isopropylthioxanthone) (Boehringer Mannheim, N 724815) and 300 ng/ml X-Gal (5-bromo-4-chloro-3 - indolyl--D-galactoside) (Bay colony, which usually contain an insert, bring on 96-alopecia microtiter plateau. In this case, each white colony placed in one nest plateau, which contains, in addition, 150 μl of LB medium with the addition of 50 μg/ml kanamycin and incubated at temperature 37oC for 20 hours to produce a mini-cultures.

Prepare the primers opposite orientation as the orientation of the gene in the vector RK not known. In this case, these primers produced by ligating nucleotide fragment described by the sequence SEQ ID No: 5, partially encoding lacticin 481, with one or another universal breakdown pUC vectors, which represent respectively the sequence of SEQ ID No: 6 or the sequence SEQ ID No: 7.

1 ál of the contents of each cell are mixed with 100 Ptolemy one of the specified primer, 6 μl of 2 mm dNTP and 2.5 µl Taq buffer (R. N. Stehelin & Cie AG, cat. No TP05b), then all the resulting mixture is put a drop Dina-Wachs (Finnzymes Oh, 02201 Espoo, Finland) (Dyna-wax, Finnzymes OS, 02201 Espoo) and heated at a temperature of 98oC for 10 minutes for lysis of bacteria; this is followed by polymerase-salewww reaction (PCR).

Next, perform gel electrophoresis, in the course of iTune clones and then extracted from these clones plasmid DNA; after that dideoxynucleotide method is sequenced DNA fragment cloned into the vector RK 19, using the kit for sequencing [Pharmacia biotech (Pharmacia Biotech), part No. 27-1682-01] and universal primers, after which based on the data characterizing the received sequence using specific primers.

In the study receive the nucleotide sequence of SEQ ID No: 2 below in the sequence listing. Mentioned nucleotide sequence that encodes the sequence of SEQ ID No: 1, which corresponds to the amino acid sequence of Mariazinha before puberty.

The following examples serve to illustrate the method of preparation and use of bacteriocin in accordance with the present invention. If there are no other reservations specified in the examples,% means weight percent.

Example 1

In BHI medium inoculant culture containing strain Micrococcus varians. Spend incubation over night at a temperature of 30oC when the swing under aerobic conditions, resulting in cultures containing 108organisms cultured strain of 1 ml of medium. Thus obtained standard the above supernatant, obtained by the method of example 1, to prepare a concentrate of the supernatant, to which is added 15 g of resin XAD-7 (Amberlite (R)). The mixture is stirred at a temperature of 4oC for 60 minutes and then filtered through filter No. 604 (Schleicher &Schuell, Germany). Then to remove all readsorbing proteins the filter is washed with 1% sodium citrate. The resin is separated and transferred into a receiving vessel containing sodium citrate and mix all the mixture for 2 minutes. After that, the resin with sodium citrate is transferred to the column and elute the sodium citrate in 50% acetonitrile and 0.1% TFU (Triperoxonane acid). The eluate is evaporated and the residue resuspended in 50 mm phosphate buffer at a pH of 6.8.

Example 3

Under cultivation on WN environment over night at a temperature of 30oC when the swing under aerobic conditions receive a 10 l culture of a strain of Micrococcus varians. Then directly to the culture add 200 g of the resin XAD-7 (Amberlite) and all gently stirred at a temperature of 4oC for 1 hour. After that, the mixture is filtered through a filter No. 604 (Schlelcher & Schuell, Germany), and the resin remaining on the filter, washed with 10 l of 50 mm acetic acid solution, pH 5.2, to remove bacteria. After that, the resin add 450 ml, stergis obtaining powder, containing variations of the present invention, which can be further used in the food industry.

Using the previously described agar test determine the bactericidal activity of the obtained powder which was previously diluted with water. Powder active not less than 105UE/g of powder.

And, finally, the above powder is added in a dose of 0.5 g/kg to obtain a traditional way of meat pins. In the cold foam contains 50 ye bacteriocin per 1 g of foam, and this amount is sufficient for complete inhibition of the development of pathogenic bacteria, including Clostridia

Example 4

This example relates to the production of moisturizers for skin care that contains the powder described in example 3, at a dose of 0.05 g/kg, which represents variation capable in activity 5 UE/g inhibit the unwanted skin bacteria such as Staphylococcus aureus and Streptococcus pyogenes.

To prepare the specified environment mixed lipid components of phase a and heat at a temperature of 75oC. Prepare the lipid phase B and heat at a temperature of 75oC slowly, while stirring, add the lipid phase And, tstuat approximately 25oC. Slowly at the same temperature, in the order listed below, add the components of the mixture Century.

The liquid phase A - %

G"U-6 stearate, glycerin and PEG-20-cetyl ether (PEG-polyethylene glycol) - 15

Liquid paraffin - 5

The oil of wheat germ, stabilized with 0.1% phenylindane (antioxidant) and 1% soybean phospholipid (see EP94109355.1) - 3

The sweet almond oil - 2

Cetyl alcohol - 1

Isostearyl isostearate - 2

2-Octyldodecyl myristate - 1

Lanolin wax - 1

The aqueous phase B - %

Methylisothiazolinone - 0,1

Demineralized water - 59,6

Placental human protein - 2

In the Additive - %

Propylene glycol and calendula extract - 2

50% soluble collagen in demineralised water to 5.8

Perfume perfume - 0,3

2.5% powder bacteriocin obtained by the method of example 3, in demineralised water - 0,2

Example 5

Described in example 3, the powder bacteriocin add in the amount of 0.5 g/kg to the means for rinsing the mouth. The tool acquires as a result, the ability to inhibit the development of pathogenic bacteria, in particular Streptococcus sorbinus. Streptococcus sanguis, Streptococcus mutans and Actinomyces viscosus in the mouth.

1. The bacteriocins from Micrococcus varians described amino acid sequence of SEQ ID No: 1.

2. The bacteriocins under item 1 for food, cosmetics or pharmaceutical products, in particular for use in dermatology and oral hygiene.

3. The DNA fragment encoding the bacteriocins under item 1, with nucleotide sequence SEQ ID No: 2.

4. The DNA fragment under item 3, in which the nucleotides with 88 228 encode a signal peptide with secretively the bacteriocins.

5. The DNA fragment under item 3, in which the nucleotides with 154 228 encode secretory bacteriocins.

6. The DNA fragment under item 3, in which the nucleotides with 88 153 encode the signal peptide.

7. The signal peptide from Micrococcus varians, encoded by nucleotides with 88 153 in the sequence SEQ ID No: 2.

8. Strain Micrococcus varians CNCM 1-1586-producer of bacteriocin under item 1.

9. The strain under item 8 for receiving food products, cosmetics or pharmaceutical products, in particular, for use in dermatology and oral hygiene.

10. Strain Micrococcus varians CNCM 1-1587-producer of bacteriocin under item 1.

12. The method of obtaining bacteriocin under item 1, comprising the cultivation of a strain of Micrococcus varians in the environment and the conditions favorable for its growth, to achieve the concentration of microorganisms in the culture medium from 107up to 1011microorganisms/ml, centrifuging the resulting culture and obtaining the extract supernatant containing bacteriocins.

13. The method according to p. 12, characterized in that the cultivated strain on p. 8 or 10.

14. The method according to p. 12, characterized in that the extract is used for food, cosmetics or pharmaceutical products, in particular, for use in dermatology and oral hygiene.

 

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