Medication (versions), composition (versions) and application of medication (versions) for reduction of halitosis

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to versions of medication for reduction of halitosis, versions of compositions based on said medications and versions of application of said medications. Device for reduction of halitosis represents strains of microorganisms Lactobacillus acidophilus, selected from group Lactobacillus acidophilus DSM 19825, Lactobacillus acidophilus DSM 19826 and Lactobacillus acidophilus DSM 19827. Version of said medication represents culture supernatant of said strains. Also claimed are compositions, which contain said microorganisms, and their application for reduction of halitosis.

EFFECT: invention makes it possible to considerably reduce concentration of peptides in saliva, depleting in this way substrate, used by anaerobic microorganisms of oral cavity microflora, which are halitosis-causing agents.

10 cl, 7 dwg, 19 ex

 

The present invention relates to a microorganism belonging to the group of lactic acid bacteria, which can significantly reduce the concentration of peptides in saliva, thereby depleting the substrate used by anaerobic microorganisms microflora of the oral cavity, which are agents that cause bad breath. Moreover, the described microorganism belongs to the group of lactic acid bacteria, which are able to stimulate the growth of Streptococcus salivarius, but does not stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis.

The present invention also relates to compositions containing the above-mentioned microorganisms, their use for the prevention and/or treatment of bad breath and/or halitosis, and ways of prevention and/or treatment of bad breath and/or halitosis.

A common problem in the field of hygiene is chronic bad breath (bad smell). A widespread method for the treatment of halitosis is in disguise or neutralize the bad odor through the use of mouthwash for mouth or chewing gum that contain, for example, menthol. However, these methods are only effective for a short period and are not effective for the long term. Therefore, there is a need for methods of prevention or treatment of halitosis, help them for a long period. This problem was solved in the prior art in various ways, all of which to a greater or lesser extent, are committed to reducing the number of anaerobic bacteria that produce volatile sulfur compounds" (LSS), such as hydrogen sulfide and methyl mercaptan, for example.

One described method for reduction of these bacteria is to remove plaque from the tongue with a scraper, to remove from the language of the substrates for bacteria. Another way is to treat language metal salt such as zinc chloride, or disinfectant, such as alcohol or chlorhexidine. However, the drawback of these methods is that the metal salt and disinfectant also inhibit the growth of other harmless or even beneficial microorganisms of the oral cavity.

One approach to the treatment and prevention of halitosis, which has been described, is to maintain the pH of saliva to a physiologically normal level. It is known that species of microbes associated with tooth decay and infections of the mucous membranes, favorable to the development of an acidic environment, types of microbes associated with the development of periodontal disease, favor the development of pH above normal, while the varieties of microbes associated with good health of your mouth, favor the development of neutral is pH. Compositions containing probiotic bacteria (such as Lactobacillus and Streptococcus)that use this mechanism are disclosed in patent applications U.S. US 200707137 and US 2006018843. International patent application W0 2007/077210 discloses a method of restoring oral microflora associated with good health of your mouth, which uses probiotics, poorly producing or not producing acid selected from the group rapidly colonizing bacteria of the oral cavity, which are normally present in the healthy microflora of the oral cavity (for example, Streptococcus (oralis), Eubacterium, Neisseria, Veilonea) in combination with substances having a pH-raising or pH-buffering agents (e.g., bicarbonate, urea, phosphates, proteins and/or salt). Application for U.S. patent US 2006045870 reveals live lactic acid bacteria belonging to the genus Weisella, which inhibit the growth of LSS-producing bacteria by interacting with them and generating hydrogen peroxide under aerobic and anaerobic conditions. Application for U.S. patent US 2006171901 discloses another method of suppressing the growth of aerobic bacteria, especially bacteria, causing helicos, which includes BPIV (bacteriocins-like inhibiting substances) - producing strains of Streptococcus salivarius and their extracts.

The lack of methods available for the prevention and treatment of halitosis, is that most of this is x way inhibit not only the growth of LSS-producing bacteria, which are the main cause of bad breath, but they also inhibit the growth of other harmless microorganisms of the oral cavity.

Therefore, the present invention is to provide alternative means and methods for the prevention and/or treatment of bad breath and/or halitosis.

Accordingly, the first object of the present invention relates to a microorganism belonging to the group of lactic acid bacteria, characterized by the fact that exhibits the following property (b), when subjected to the following analysis:

Analysis (a):

(a) the microorganism is cultivated for 24 hours at 37°C under anaerobic conditions in an artificial medium containing 15 g/l of peptides with the initial density of cells 1·107cells/ml;

(b) cells are removed by centrifugation at 4000·g for 15 min; and

(C) determine the concentration of the peptides in the resulting supernatant.

Property (b):

The microorganism leads to a decrease in the concentration of peptides in the culture medium so that the concentration of peptides in the supernatant after incubation for 24 hours reduced at least by 20% in comparison with the initial concentration of 15 g/l, i.e. the microorganism is able to reduce the concentration of peptides in the environment in the analysis (a)at least 20%.

In a preferred embodiment, the image is placed microorganism according to the invention additionally exhibits the following property (B), when subjected to the following analysis:

(a) the microorganism is cultivated in 100 ml of artificial medium at 37°C for 24 hours under anaerobic conditions, with an initial density of cells 1·107cells/ml;

(b) the cells are then centrifuged at 4000·g for 15 min and resuspended in 20 ml of N2O;

(b) 10 mg of lyophilised bacteria resuspended in N2O and centrifuged at 4000·g for 10 min;

(d) 1 ml of artificial medium containing 7 g/l of peptides added to the precipitate, cells resuspended in the environment and after 5 min of aerobic incubation at 37°C. cells are removed by centrifugation at 4000·g for 15 min; and

(d) determine the concentration of the peptides in the resulting supernatant environment.

Property (B): lyophilized bacteria lead to a decrease in the concentration of the peptides in the resulting supernatant medium, at least 20% compared with the concentration of the medium at the beginning of the incubation period (7 g/l).

In a preferred embodiment of the invention, the concentration of peptides in the assay (a) or (A) reduced by the use of a microorganism according to the invention, at least 30%, more preferably at least 40% and even more preferably at least 50%.

In a particularly preferred embodiment of the invention the microorganism according to the invention is capable red eye reduction shall be the concentration of peptides in the analysis (A), at least 60% and even more preferably at least 70%.

Thus, the present invention provides a microorganism which can effectively reduce the concentration of peptides in its environment, in particular also in saliva, as it is described in the accompanying examples. As you know, bad breath is caused by a phenomenon in which the relationship between the healthy flora of the oral cavity (formed mainly Streptococcus salivarius) and pathogenic flora of the oral cavity (formed mainly anaerobic gram-negative bacteria) is shifted in the direction of anaerobic gram-negative bacteria, which decompose the proteins present in the saliva, to volatile compounds. This leads to the formation of volatile sulfur compounds that cause bad breath.

The microorganism of the present invention is able to reduce bad breath by reducing the number of peptides and, consequently, depletion of substrate anaerobic gram-negative bacteria flora of the oral cavity.

The term "artificial environment" refers to a chemically defined environment, i.e. the environment, the chemical composition of which is known. An artificial environment can be any artificial environment suitable for culturing this organism. In one preferred embodiment, izaberete the Oia artificial environment is such an artificial environment, as is disclosed in U.S. patent US 6,340,585.

In a preferred embodiment of the invention the artificial medium is a medium with the following composition:

Guanine:0.1 g/l
Cytosine:0.1 g/l
Thymidine:0.1 g/l
2'-Deoxyadenosine:0.1 g/l
2'-Deoxyuridine:0.1 g/l
To2NRA4:2 g/l
Sodium acetate:5 g/l
gSO4-heptahydrate:0.1 g/l
Diammonium hydrocitric:2 g/l
l2-dihydrate:0.5 g/l
Oleic acid:0,1% (weight / volume)
Cyanocobalamin:0.02 mg/l
Riboflavin:10 mg/l
Folic acid:0,2mg/l
Pyridoxal-5-phosphate monohydrate:10 mg/l
4-Aminobenzoic acid:0.2 mg/l
D (+)-Biotin:1 mg/l
Ascorbic acid:500 mg/l
Nicotinic acid:10 mg/l
CA-Pantothenate:10 mg/l
Thiamine:1 mg/l
Cobalt(II)-Nitrate-Uranyl:500 mg/l
nSO4-monohydrate:20 mg/l
nSO4-heptahydrate:500 mg/l
Na2Moo4:0.04 mg/l
Extract PTU (Ohiy, Deutsche15 g/l (or as specified in this
Hefewerke, Germany):case)
D-Glucose monohydrate:10 g/l

The term "containing 15 g/l of peptides" (or "containing 7 g/l of peptides") means that the art is owned by the environment at the beginning of the incubation period contains 15 g/l of peptides (or 7 g/l peptides, respectively). In principle, the peptide can be any peptide. In a preferred embodiment of the invention the peptides contained in an artificial environment, are in the form of yeast extract, preferably an extract of PTU. Extract PTU (peptide ultrafiltrating extract) can be purchased in Ohly, Deutsche Hefewerke, Germany. This ultrafiltrating nizkosolevaya yeast extract with a high content of readily available peptides, and preferably it has the following characteristics:

Analysis:

Dry material:96%
Protein (N x 6,25) in dry material:72,9%
Full nitrogen in dry material:11,7%
NaCl:≤ 1,0%
Ash:10%
pH (2% solution):the 5.7

Vitamins (typical):

TakingITGlobal×HCl (B1):1.2 mg/100 g
Riboflavin (B2):7,0 mg/100 g
Pyridoxine×HCl (B6): 5.9 mg/100 g
Nicotinic acid:to 47.8 mg/100 g
Biotin:0,022 mg/100 g
Ca-D-Pantothenate:17.9 mg/100 g
Folic acid:3.7 mg/100 g
Amino acid profile (typical):as shown in Figure 7

The term "initial cell density in cells/ml" means that artificial environment inoculums in the early period of cultivation of the microorganism, so that in the medium was 1·107cells/ml

The concentration of peptides can be determined by any known specialist in this field by the way. Well-studied methods are, for example, biuret method, the method of Lowry, the method of Bradford. Moreover, you can use any commercially available kit or other system for determining the concentration of peptides. One preferred example is the system or sets based on fluorescent dyes, such as a set of Quant-it Protein company Invitrogen.

The decrease in the concentration of peptides in the above tests (a) and/or (A) preferably is investigated as described in the accompanying Examples.

As is provided in the accompanying Examples, unexpectedly, it was found that lactic acid bacteria can exhibit the ability to reduce significantly in their environment the concentration of peptides. This effect manifests itself not only with live bacteria, but also with their liofilizovannye forms. Moreover, the Examples show that the microorganisms according to the invention exhibit the above effect, not only in the above analyses, but also in saliva, and that the presence of the microorganism according to the invention leads to a significant reduction in the production of H2S when you add in the saliva.

In particular, in the preferred embodiment of the invention the microorganism according to the invention also exhibits the following property (g)when subjected to the following analysis (in):

Analysis of (in):

(a) the microorganism is cultivated in 100 ml of artificial medium at 37°C for 24 hours under anaerobic conditions, with an initial density of cells 1·107cells/ml;

(b) the cells are then centrifuged at 4000·g for 15 min and resuspended in 20 ml of N2About;

(C) the cells are then frozen to -80°C and lyophilizers in vacuum for 16 hours;

(d) 10 mg of lyophilised bacteria resuspended in H2O globalinvacom tablet and centrifuged at 4000·g for 10 min;

(d) 1 ml of artificial medium containing 3 g/l of peptides added to the precipitate, the image is of valemus after centrifugation, after 5 min incubation at 37°C. cells are removed by centrifugation at 4000·g for 15 min;

(e) the supernatant is then transferred into a new Globaloney tablet and then inoculant from 10 to 100 μl, preferably 50 ál, non-sterile human saliva and incubated under anaerobic conditions for 6 hours at 37°C, while Globaloney tablet cover with a sterile filter paper impregnated with lead acetate;

(g) producing microorganisms of hydrogen sulfide in the reaction is controlled by tracking the blackening of the filter paper.

Property (d): in the presence of the microorganism according to the invention, the blackening of the filter paper impregnated with lead acetate, is reduced compared with the control, in which the environment was not pre-incubated with the specified microorganism. Reduced blackening of the filter paper is an indicator of reduced production of H2S by bacteria in sterile human saliva used for inoculation of the medium.

The term "reduced production of H2S" means the reduction of the production of H2S, at least 10%, more preferably at least 20%, even more preferably at least 30% and especially preferably at least 40% or even at least 50% compared to what stralem. The reduction can be measured, for example, using densitometric analysis of the blackening of the filter paper. Alternatively, the production of hydrogen sulfide in steps (e) and (f) not determined using the filter paper, as measured by headspace analysis using gas chromatography.

The second object of the present invention also relates to a microorganism belonging to the group of lactic acid bacteria characterized in that they are able to stimulate the growth of Streptococcus salivarius, but not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis.

The term "encourage" the growth of microorganisms of the species Streptococcus salivarius means that the growth of these microorganisms is increased by contact with a microorganism according to the invention. Increased growth preferably means an increase in proliferation, i.e. cell divisions per unit time. Alternatively, the term "stimulate" also refers to the growth of individual cells in size. The size of bacterial cells can be assessed using flow cytometry (e.g., flow cytometer Becton-Dickinson FACSort, San Jose, CA) after staining with the dye SYBR Green I (Molecular Probes, USA). The size of bacterial cells evaluate mode Side-Angle Light Scatter (SSC). Increased growth, thus, increase the production of biomass per unit time.

With kalinovaya growth of the corresponding microorganism preferably can be observed in vitro, more preferably, in the analysis, in which the microorganism according to the invention in contact with Streptococcus salivarius and is determined by the growth of Streptococcus salivarius. Growth can be determined by counting the number of cells/colonies after different time intervals of incubation and compared with a control that does not contain a microorganism according to the invention, thereby allowing them to know whether the increase in growth.

Analysis of in vitro to determine the stimulation of growth described in the Examples and contains the so-called "Photometric coincubation analysis. Briefly, this analysis contains the following steps:

(a) a microorganism belonging to the group of lactic acid bacteria to be tested are mixed with Streptococcus salivarius in the ratio of cells 1:100 (lactic acid bacteria: Streptococcus salivarius)1/2triticosecale medium with yeast extract ('TSY medium, trypticase soy yeast extract);

(b) the culture suspension is incubated under aerobic conditions for 12 hours at 37°C;

(C) as a control use unspent1/2environment'TSY or light environment MRS;

(g) determine the maximum optical density (OD600,maxand/or determine the maximum growth rate (Vmaxduring exponential growth; and

(d) the microorganism is classified as a microorganism capable stimuleren the th growth of Streptococcus salivarius, if the maximum optical density (OD600, maxand/or the maximum growth rate (Vmaxincreases of at least 10% compared to control.

The term "1/2environment'TSY" refers to'TSY environment, which is diluted with N2O in the ratio 1:1 (by vol.: vol.).

In a preferred embodiment of the invention, the incubation is carried out in 96-well pad. In another preferred variant of the invention, the incubation is carried out in a microplate spectrophotometer, Bio-Tek PowerWave (Biotek Instruments Gmbh, Germany).

Preferably OD600, maxand Vmaxis determined as follows.

The optical density OD at 600 nm is measured over an extended period of time, preferably about 8 to 12 hours after the start of incubation at uniform intervals, for example every 2.5 minutes. To determine OD600, maxpreferably spend incubation for 10 hours.

To determine the value OD600, maxcalculate the average of the three highest measured values.

The value of Vmaxpreferably define, choosing 15 consecutive values, which show the steepest gradient. Unit, showing the Vmaxis mos/min (units ellipticheskoi density per minute). Definition OD600, maxto calculate the Vmaxpreferably is conducted over a period of time, allows you to reach the exponential phase of growth kultuuriruumis microorganism.

Preferably the microorganism according to the invention leads to an increase in the maximum optical density (OD600, max) or the maximum growth rate (Vmax) Streptococcus salivarius in the above analysis, at least 15%, more preferably at least 20%, even more preferably at least 30% and especially preferably at least 40%, 50%, 60%, 70% and even 80% compared to control.

In a preferred embodiment of the invention described above, the microorganism according to the invention not only stimulates the growth of Streptococcus salivarius, but also stimulates the growth of at least one other microorganism healthy microflora of the oral cavity. Examples of such pathogens are Streptococcus oralis and Streptococcus epidermidis. Stimulation of these bacteria can be measured using the above analysis.

The above-described microorganism according to the invention is also characterized by the fact that it does not stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis. The microorganism is not stimulating the growth of the microorganism transient pathogenic micro flora, if it does not lead to increased growth of Streptococcus mutans and/or Porphyromonas gingivalis in contact with them. Stimulation of growth or lack of it can be explored in vtro. The in vitro assays for determination of stimulating growth or lack thereof is described in the Examples and contains the so-called "Photometric coincubation analysis. In short, this analysis in the case of Streptococcus mutans contains the following steps:

(a) a microorganism belonging to the group of lactic acid bacteria, which will be tested, cultivated under anaerobic conditions in 96-well-plate with 150 μl of the artificial medium for 24 hours at 37°C, cells precipitated by centrifugation at 4000·g for 15 min and recover the supernatant;

(b) Streptococcus mutans cultured under anaerobic conditions in 5 ml'TSY environment in closed 15-ml Falcon tubes overnight at 37°C;

(C) culture of cells of Streptococcus mutans are mixed in a volume ratio of 2:1 with the supernatant from step (a);

(g) the culture suspension is incubated under aerobic conditions for 12 hours at 37°C;

(d) quality control use unspent1/2environment'TSY or light environment MRS;

(e) determine the maximum optical density (OD600, maxand/or determine the maximum growth rate (Vmaxduring exponential growth; and

(g) the microorganism is classified as a microorganism is unable to stimulate the growth of Streptococcus mutans, if the maximum optical density (OD600, maxand/or maximum rate of growth is (V max) is not increased compared to control.

Alternatively, such analysis may include the following steps:

(A) the tested microorganism belonging to the group of lactic acid bacteria, mixed with Streptococcus salivarius in the ratio of cells 1:100 (lactobacillus: S. mutans) in1/2environment'TSY;

(B) the culture suspension is incubated under aerobic conditions for 12 hours at 37°C;

(B) as a control use unspent1/2environment'TSY or light environment MRS;

(G) determine the maximum optical density (OD600, maxand/or determine the maximum growth rate (Vmaxduring exponential growth; and

(D) the microorganism is classified as a microorganism is unable to stimulate the growth of Streptococcus mutans, if the maximum optical density (OD600, maxand/or the maximum growth rate (Vmax) is not increased compared to control.

In the case of Porphyromonas gingivalis analysis contains the following steps:

(C) the test microorganism belonging to the group of lactic acid bacteria, kulturyrussia under anaerobic conditions in 96-well-plate with 150 μl of the artificial medium for 24 hours at 37°C, cells precipitated by centrifugation at 4000·g for 15 min and recover the supernatant;

(and) Porphyromonas gingivalis grown under anaerobic conditions in 5ml medium FAB in closed 15-ml Falcon tubes overnight at 37°C;

(K) cell culture Porphyromonas gingivalis are mixed in a volume ratio of 2:1 with the supernatant from step (C);

(l) the culture suspension is incubated under aerobic conditions for 45 hours at 37°C;

(m) quality control use unspent Wednesday FAB;

(h) determine the optical density (OD600after 10, 15, 21, 39, and 45 hours of incubation; and

(a) the microorganism is classified as a microorganism is unable to stimulate the growth of Porphyromonas gingivalis, if the optical density (OD600in every moment of measurement is not increased compared to control.

Alternatively, this analysis contains the following steps:

(C) the test microorganism belonging to the group of lactic acid bacteria, mixed with Porphyromonas gingivalis in the ratio of cells 1:100 (lactobacillus: P. gingivalis) in the FAB environment;

(And) the culture suspension is incubated under aerobic conditions for 45 hours at 37°C;

(K) as a control use unspent Wednesday FAB;

(L) determine the maximum optical density (OD600, maxand/or determine the maximum growth rate (Vmaxduring exponential growth; and

(M) the microorganism is classified as a microorganism is unable to stimulate the growth of Porphyromonas gingivalis, if the maximum optical density (OD600, maxand/or the maximum growth rate (Vmax) not HC is growing compared to control.

In a preferred embodiment of the invention the incubation in steps (g) and (B) is carried out in a 96-well pad. In another preferred variant of the invention, the incubation is carried out in a microplate spectrophotometer, Bio-Tek PowerWave (Fa Biotek Instruments GmbH, Germany).

In a preferred embodiment of the invention the incubation steps (l) and (I) is carried out in a 96-well pad. In another preferred variant of the invention, the incubation is carried out in an anaerobic workstation Whitley DG250 (Meintrup-DWS, Germany).

In relation to OD600, maxand Vmaxapply the same thing that has been formulated here above.

The microorganism is not stimulating the growth of the microorganism Streptococcus mutans or Porphyromonas gingivalis, if growth does not increase or only slightly increased upon contact with the microorganism. "Slightly increase" means the increase by no more than 5% compared to control, more preferably not more than 2% compared with the control. The term "not increasing" means that there can be found no statistically significant differences between the growth of Streptococcus mutans or Porphyromonas gingivalis in contact with the microorganism of the invention in comparison with the control, where no microorganism of the invention. The term does not increase in a preferred variant is NTE embodiment of the invention also includes those cases when the microorganism actually reduces the growth of Streptococcus mutans or Porphyromonas gingivalis, i.e. it inhibits the growth of this microorganism.

In another preferred embodiment of the invention, the microorganism of the present invention is not adversely affected by the growth of Streptococcus mutans or Porphyromonas gingivalis. The term "do not be adversely affected" means that there can be found no inhibition of growth of Streptococcus mutans or Porphyromonas gingivalis in contact with the microorganism of the invention in comparison with the control, where no microorganism of the invention.

In a preferred embodiment of the invention the microorganism according to the invention does not stimulate not only the growth of Streptococcus mutans and/or Porphyromonas gingivalis, but also does not stimulate the growth of at least one other pathogen microflora of the oral cavity. Representatives of pathogenic bacteria of the oral cavity are anaerobic, gram-negative bacteria. Other examples are Actinobacillus actinomycetemcomitans, Actinomyces naeslundii, Fusobacterium nucleatum, Fusobacterium nucleatum polymorphum, Prevotella intermedia, Solobacterium moorei, Streptococcus gordonii, Streptococcus mitis, Streptococcus sanguinis, Tannerella forsynthensis and Treponema denticola.

Stimulation or lack of stimulation of the growth of these bacteria can be measured using the assays described above for S. Mutans and P. gingivalis.

In a preferred embodiment of the invention described in the above microorganisms according to the invention are characterized by the fact that they not only show the effect of stimulating the growth of Streptococcus salivarius in the form of living cells, but also in the culture supernatant. This means that also the culture supernatant obtained from a microorganism according to the invention, showing the effect of stimulating the growth of Streptococcus salivarius. Preferably, this effect is manifested in the following analysis:

(a) Streptococcus salivarius cultivated under anaerobic conditions in a 6-hole tablets with 8 ml'TSY medium over night at 37°C;

(b) the tested microorganism belonging to the group of lactic acid bacteria, cultivated under anaerobic conditions in 96-well tablets with 150 μl of the artificial medium for 24 hours at 37°C, cells precipitated by centrifugation at 4000·g for 15 min and the supernatant restore;

(C) cell culture Streptococcus salivarius from step (a) are mixed in a volume ratio of 2:1 to 4:1 with the supernatant from step (b)1/2environment'TSY;

(g) the culture suspension is incubated under aerobic conditions for 12 hours at 37°C;

(d) quality control use unspent1/2environment'TSY or light environment MRS;

(e) determine the maximum optical density (OD600, maxand/or determine the maximum growth rate (Vmaxduring exponential growth; and

(g) the microorganism is classified as a microorganism, SP is capable to stimulate the growth of Streptococcus salivarius, if the maximum optical density (OD600, maxand/or the maximum growth rate (Vmaxincreases of at least 10% compared to control.

In a preferred variant of the invention, the incubation is carried out in a 96-well pad. In another preferred variant of the invention, the incubation is carried out in a spectrophotometer for microplate Bio-Tek PowerWave (Fa Biotek Instruments GmbH, Germany).

In relation to OD600, maxand Vmaxapply the same thing that has been formulated here above.

Preferably the microorganism according to the present invention leads to an increase in the maximum optical density (OD600,max) or the maximum growth rate (Vmax) Streptococcus salivarius in the above analysis, at least 15%, more preferably at least 20%, even more preferably at least 30% and especially preferably at least 40%, 50%, 60%, 70% or even 80% compared to control.

In a particularly preferred embodiment of the invention stimulate the growth of Streptococcus salivarius shown by the microorganism according to the invention, is resistant to heat treatment, i.e. it also occurs when cells (or their extracts) or culture supernatant subjected to heat treatment. Heat treatment of CR is doctitle is a thermal processing at a temperature between 60°C and 100°C, more preferably between 70°C and 90°C, even more preferably between 75°C and 85°C and most preferably at a temperature of about 80°C or exactly 80°C.

Typically, the heat treatment should last for at least 1 minute. Preferably, the heat treatment lasts for at least n minutes, where n is an integer in the range from 2 to 60, where n=10, or 15, or 20 is particularly preferred. However, in principle there is no upper limit for the time of incubation. However, preferably it should not be more than 4, 3, 2 or 1 hour. The most preferred heat treatment is the treatment for 10 minutes at a temperature of 80°C in the incubator. The most preferred heat treatment is a treatment that destroys any protein function and any function of the viability of the cells, and thus, the above mentioned microorganism belonging to the group of lactic acid bacteria, is distinguished from another microorganism that he is still able to stimulate the growth of Streptococcus salivarius. Therefore, it is very useful for use in any food product, food product, beverage, or compositions in the context of the present invention, if it is desirable that the microorganism was not alive.

After cooling ability of the microorganism according to the image the structure (or its extracts or culture supernatants to stimulate the growth of Streptococcus salivarius is determined in the analysis, as described herein above or as described in the accompanying Examples. In relation to culture supernatant of a microorganism according to the invention corresponding to the analysis preferably includes the following steps:

(C) Streptococcus salivarius cultivated under anaerobic conditions in a 6-hole tablets with 8 ml'TSY medium over night at 37°C;

(and) the tested microorganism belonging to the group of lactic acid bacteria, cultivated under anaerobic conditions in 96-well tablets with 150 μl of the artificial medium for 24 hours at 37°C, cells precipitated by centrifugation at 4000·g for 15 min and the supernatant restore;

(K) the supernatant is incubated at 80°C for 10 min in the incubator and then cooled to room temperature;

(l) the cell culture Streptococcus salivarius from step (C) are mixed in a volume ratio of 2:1 with the supernatant from step (K) in1/2environment'TSY;

(m) the culture suspension is incubated under aerobic conditions for 12 hours at 37°C;

(h) quality control use unspent1/2environment'TSY or light environment MRS;

(a) determine the maximum optical density (OD600, maxand/or determine the maximum growth rate (Vmaxduring exponential growth; and

(p) the microorganism is classified as a microorganism capable of stimulating the growth of Streptcoccus salivarius, if the maximum optical density (OD600, maxand/or the maximum growth rate (Vmaxincreases of at least 10% compared to control.

Moreover, also the property of a lack of stimulating the growth of Streptococcus mutans and/or Porphyromonas gingivalis bacterium according to the invention is resistant to heat treatment. In relation to determining the time of heat treatment is applied the same as was set forth above.

In another preferred embodiment of the invention stimulate the growth of Streptococcus salivarius shown by the microorganism according to the invention is resistant to freeze-drying, i.e. it also occurs when cells are exposed to liabilitiesa processing. Liabilitiesa processing preferably represents lyophilizers processing, in which cells (or their extracts) or cell supernatant is first frozen to -80°C and then lyophilizer in vacuum for 16 hours. After liabilitiesa processing ability of the microorganism according to the invention to stimulate the growth of Streptococcus salivarius may be tested using the tests already described above, or as described in the accompanying Examples. In relation to culture supernatant of a microorganism according to the invention corresponding to the analysis preferably includes the following steps:

(R) Streptococcu salivarius cultivated under anaerobic conditions in a 6-hole tablets with 8 ml'TSY medium over night at 37°C;

(C) the test microorganism belonging to the group of lactic acid bacteria, cultivated under anaerobic conditions in 96-well tablets with 50 μl of the artificial medium in closed 100 ml bottles over night at 37°C, cells precipitated by centrifugation at 4000·g for 15 min and the supernatant restore;

(t) 20 ml of the supernatant from step (C) is frozen to -80°C and lyophilizers in vacuum for 16 hours;

(I) freeze-dried supernatant resuspended in 20 ml of N2About;

(f) cell culture Streptococcus salivarius from step (p) are mixed in a volume ratio of 2:1 with the supernatant from step (I)1/2environment'TSY in 96-well tablets;

(x) the culture suspension is incubated under aerobic conditions for 12 hours at 37°C;

(h) quality control use unspent1/2environment'TSY or light environment MRS;

(III) determine the maximum optical density (OD600, maxand/or determine the maximum growth rate (Vmaxduring exponential growth; and

(y) the microorganism is classified as a microorganism capable of stimulating the growth of Streptococcus salivarius, if the maximum optical density (OD600, maxand/or the maximum growth rate (Vmaxincreases of at least 10% compared to control.

According to especially preferred is the option of carrying out the invention the microorganism according to the invention exhibits both properties, accordingly described for the first and second object of the present invention, i.e. it shows the properties (b) and/or (B)as described for the first object (a sharp decrease in the concentration of peptides) and exhibits properties as described for the second object (stimulation of growth of Streptococcus salivarius and lack of stimulating the growth of Streptococcus mutans and/or Porphyromonas gingivalis).

The microorganisms according to the invention as described here above, due to their properties allow them to shift the balance of the microflora of the oral cavity in the direction of Streptococcus salivarius, which leads to improvement from the point of view of the development of fewer unpleasant mouth odor.

As apparent from the above, all the above features make the above mentioned microorganism belonging to the group of lactic acid bacteria, a suitable agent for lowering unpleasant mouth odor and/or halitosis or for the prevention and/or treatment of bad breath and/or halitosis, is particularly unpleasant mouth odor and/or halitosis caused by pathogenic microorganisms microbial flora of the oral cavity, especially anaerobic gram-negative bacteria. Accordingly, the microorganism according to the invention has an effect to reduce unpleasant mouth odor and thus is a useful agent for the prevention and/or treatment of bad breath and/or x is licosa.

The term "preventing unpleasant mouth odor" includes the prevention of unpleasant mouth odor. Accordingly, a subject that has never been faced with these microorganisms, which are responsible for the development of unpleasant mouth odor, but may have a chance encounter with them, i.e. to be infected with such organisms, or entity that has a well-balanced microflora of the oral cavity, benefits from the use of, for example, microorganisms, compositions, uses and methods of the present invention, in this case, the specified subject will not suffer from unpleasant mouth odor. Therefore, microorganisms, compositions, applications and methods of the present invention may, for example, be used by older children and younger children or young animals to prevent bad smell from the mouth, as the mouth of children and young animals in the norm is free from microorganisms, responsible for the development of unpleasant smell from a mouth. However, microorganisms and compositions used in accordance with the present invention is not limited to the appointment of younger and older age or young animals.

The terms "treatment of bad breath and treatment of halitosis include the introduction of micro-organisms or compositions described herein, su is yuktam, suffering from bad breath and/or halitosis, to reduce the amount of produced unpleasant odor.

If desired, the microorganism according to the present invention is a probiotic microorganism, which in addition to the effect of reducing unpleasant mouth odor has a beneficial effect on the host organism into which it is injected. "Probiotic", according to the generally accepted definition is "a lively addition to food, which is beneficial to an animal host by improving its microbial balance in the gut.

Accordingly, the present invention provides the use of easy to introduce bacteria, which are suitable organisms for use in food in addition to the effect of reducing the unpleasant smell from a mouth can be useful as probiotics.

Surprisingly, the effect of microorganisms according to the invention effectively remove peptides from the environment and, thus, also of saliva, thereby preventing other microorganisms present in the microflora of the oral cavity that are responsible for producing substances, leading to the formation of unpleasant mouth odor, to develop appropriate substances can also be observed with inactivated forms of microorganisms, such as lyophilized form or shape, there is s in the processing of UV-radiation or radiation.

Most importantly, the effect is also observed in the presence of saliva, which makes the microorganism according to the invention particularly suitable for use in oral form or as an additive in food, feed or drinks. Amazingly, thermally inactivated or lyophilized form, especially equivalents, derivative, or fragment(s) of these microorganisms disclosed here, is still capable of specifically effectively reduce the concentration of peptides in the above-described assays.

Similarly, also the property of the microorganism according to the present invention, described for the second object of the present invention, i.e. to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis, is manifested not only by the microorganisms themselves, but also with the culture supernatant of a microorganism and inactivated forms. In particular, the property of stimulating the growth of Streptococcus salivarius and lack of stimulating the growth of Streptococcus mutans and/or Porphyromonas gingivalis is resistant to heat treatment and is resistant to liabilitiesa processing.

These amazing effects to give advantages to the use of these inactivated forms of cultural supernatants, analogue(s) or fragment(s) of the indicated microorganisms as well as their mutants or derivatives in compositions for application the animals, preferably humans or mammals, for the prevention and/or treatment of bad breath and/or halitosis. In particular, these inactivated form, cultural supernatant, analogs or fragments can easily be added to any composition, for example a cosmetic or pharmaceutical composition, food or feed product, or drink, etc. in Addition, the production of such inactivated forms of cultural supernatants, analogues or fragments is cheap and easy and they can be stored for long periods of time without losing their ability to reduce the concentration of peptides and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis. A particular advantage of the microorganism according to the invention is that it retains its ability to reduce the concentration of peptides and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis, if it is liofilizirovannam, or spray dried, or simply dried. Moreover, the ability to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis is maintained even after the heat treatment. The above properties make the microorganism according to the invention suitable for use in the compositions, op is sledding here.

In addition, in the preferred embodiment of the invention the microorganism according to the present invention exhibits the above properties (i.e. lower concentrations of peptides and/or stimulation of the growth of Streptococcus salivarius and lack of stimulating the growth of Streptococcus mutans and/or Porphyromonas gingivalis) in the presence of saliva. Saliva is an exogenous secret secreted by salivary glands. It is a complex fluid containing in addition to about 99% water and many organic and inorganic compounds. Physiological components of saliva are, including enzymes, such as mucines, lactoferrin, Proline-rich proteins, cystatin, histamine, or Stateline, or soluble lg. Thus, although saliva has many potentially interfering substances, the above-mentioned properties of the microorganism according to the invention does not preclude the presence of saliva.

The above characteristics of the above mentioned microorganism belonging to the group of lactic acid bacteria, make it a reliable and effective agent for prevention and/or treatment of bad breath and/or halitosis as it is mainly introduced in various forms in the mouth, including oral cavity and teeth, where, including saliva is present, including certain protease and low pH values after proglet is of carbohydrate, contained in the food product. Moreover, the heat resistance and/or lyophilization has the best advantages of adding the above-mentioned microorganism according to the invention belonging to the group of lactic acid bacteria, as an additive to the food product during the cooking of the specified food product. Namely food often thermally sterilized, pre-cooked, pasteurized and the like, which is detrimental to the viability of the microorganisms.

Other embodiments of the invention and advantages of the invention are formulated partly here and partly may be obvious from the description or may be learned from the application of the invention.

Before the invention is described in detail, it should be understood that this invention is not limited to the particular methodology, regulated procedures, bacteria, vectors and reagents, etc. described herein, as they may vary. Also it should be understood that the terminology used here needed to just describe private embodiments of the invention and is not intended to limit the scope of the present invention, which will be limited only by the attached claims. If not defined otherwise, all technical and scientific terms, and is used here have the same values, which usually refers to an average expert in the field.

Preferably, as used herein, the terms provide definitions, as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W. Naqel, B. and Kölbl. H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout the description and the claims which follows, unless the context requires otherwise, the word "contain" and variations such as "contains" and "contain", it will be assumed enable set integer or step or group of integers or steps but not the exclusion of any other integer or step or group integer or step.

Several documents are cited throughout the text of this description. Each of the documents cited herein (including all patents, patent applications, scientific publications, technical requirements of the manufacturer, instructions and so on), or above or below, hereby incorporated by reference in its entirety. Nothing here should be construed as an assumption, that the invention does not have the right to precede such disclosure by virtue of the preceding inventions.

It should be noted that as used herein and in the appended claims, the only forms include multiple objects, unless the context clearly indicates otherwise. So what Braz, for example, reference to "a reagent" includes one or more of these various reagents, and reference to "the method" includes reference to equivalent steps and methods known to the average person skilled in the art that could be modified or substituted for the methods described here.

When used in the context of this invention, the term "microorganism belonging to the group of lactic acid bacteria" includes a microorganism(s)that(s) is(are) to bacteria, especially fermenting gram-positive eubacteria, specifically to the family lactobacteriaceae, including lactic acid bacteria. Moreover, the term also encompasses derivatives or mutants or analogs, or fragments, such as cell extracts or membrane fractions described herein specified(s) organism(s)that store the above-described property (i.e. to reduce the concentration of peptides and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis). The terms "derivative", "mutants", "analogs" and "fragments" described herein elsewhere. Lactic acid bacteria from the taxonomic point of view is divided into sections Streptococcus, Enterococcus, Leuconostoc and Lactobacillus. The above-mentioned microorganism belonging to the group of lactic acid bacteria is preferably a form of Lactobacillus. Present Italy group of lactic acid bacteria generally lack porphyrins and cytochromes, do not paired with the electron transfer phosphorylation and, consequently, receive energy only by substrate phosphorylation. That is, in lactic acid bacteria ATP is synthesized through the fermentation of carbohydrates. All lactic acid bacteria growing under anaerobic conditions, however, unlike many anaerobes most lactic acid bacteria are not sensitive to oxygen and can thus grow in his presence as well as absence. Accordingly, the above mentioned microorganism belonging to the group of lactic acid bacteria, preferably are aerotolerant anaerobic lactic acid bacteria, preferably belonging to the genus Lactobacillus.

The above-mentioned lactic acid bacteria are preferably rod-shaped or spherical, varying from long and long to short curved rods, and, moreover, are preferably fixed and/or asporogenic and produce lactic acid as a major or sole product of fermentative metabolism. The genus Lactobacillus belongs to the above-mentioned microorganism is divided on the basis of the following characteristics into three main groups, in accordance with what is envisaged that the aforementioned Lactobacillus species may belong to each of the three main subgroups:

(a) homofermentative lactic acid bacteria

(i) producing lactic acid, preferably L-, D-or DL-isomer(s) of lactic acid, in the amount of at least 85% of glucose through the path of Embden-Meyerhoff;

(ii) growing at 45°C, but not at the temperature of 15°C;

(iii) in the form of long rods; and

(iv) having clearinterval acid in the cell wall;

(b) homofermentative lactic acid bacteria

(i) producing lactic acid, preferably L - or DL-isomer(s) of lactic acid through the path of Embden-Meyerhoff;

(ii) growing at the temperature of 15°C, showing a variable growth at 45°C;

(iii) in the form of short rods or coryneform bacteria; and

(iv) having ribitol and/or clearinterval acid in the cell wall;

(C) heterofermentative lactic acid bacteria

(i) producing lactic acid, preferably DL-isomer of lactic acid, in the amount of at least 50% of the glucose through pentose-phosphate path;

(ii) producing carbon dioxide and ethanol;

(iii) showing variable growth at a temperature of 15°C or 45°C;

(iv) in the form of long or short sticks; and

(v) having clearinterval acid in the cell wall.

On the basis of the above-described characteristics of the above microorg the mechanisms can be attributed to the group of lactic acid bacteria, in particular to the genus Lactobacillus. When using classical taxonomy, for example, adhering to the appropriate descriptions in the publication "Bergey''s Manual of Systematic Bacteriology" (Williams & Wilkins Co., 1984), it may be decided to include the organism to the genus Lactobacillus. Alternatively, classification of microorganisms of the genus Lactobacillus can be carried out using methods known in this field, for example, using their metabolic maps, i.e. comparable information about the ability of such(s) organism(s) to metabolize sugar, or using other methods described, for example, Schleifer et al., System. Appl. Microb., 18 (1995), 461-467 or Ludwiq et al., System. Appl. Microb., 15 (1992), 487-501. The above-mentioned microorganisms are able to metabolize sources of sugar that is typical and well known in the field of machinery for microorganisms belonging to the genus Lactobacillus. Preferably, however, the above-mentioned microorganism has a metabolic map, selected from the group consisting of:

(i) the microorganism metabolizes D-lactose, but not L-sorbose, and/or D-sucrose, and/or D-inulin,

(ii) a microorganism metabolizes inulin,

(iii) the microorganism metabolizes L-sorbose, but not D-lactose, and/or D-sucrose, and/or inulin, and

(iv) the microorganism metabolizes L-sorbose, D-lactose and inulin.

Preferably, the above microorgani the m has a metabolic map selected from the group consisting of:

(i) the microorganism metabolizes D-lactose, but not L-sorbose, D-sucrose and inulin,

(ii) a microorganism metabolizes L-sorbose, D-lactose and inulin, but not D-sucrose

(iii) the microorganism metabolizes L-sorbose, but not D-lactose, D-sucrose and inulin, and

(iv) the microorganism metabolizes L-sorbose, D-lactose, D-sucrose, but not inulin.

Of course, the above-mentioned microorganism is not limited to metabolizirovannom sugars listed in the above diagram metabolic maps, he may be able to metabolize other sugars that are normally metabolized by Lactobacillus species.

The identity of the above-mentioned micro-organisms to the genus Lactobacillus can also be determined using other methods known in this field, for example, using SDS-PAGE gel electrophoresis of total protein specific types of microorganisms, belonging to which you want to define, and then comparing them with known and characterized strains of the genus Lactobacillus. The above methods in order to prepare a profile of total protein, as well as quantitative analysis of these profiles well-known specialist in this field. However, the results are quite reliable, because each stage of the process is fairly standardized. Faced with the requirement of precision when amnestyinternational to the genus Lactobacillus, standardized methods are constantly communicated to the public by their authors, as did the authors of the Pot, etc. during the seminar, organized by the European Union, at the University of Ghent, Belgium, 12-16 September, 1994 (Fingerprinting techniques for classification and identification of bacteria, SDS-PAGE of whole cell protein). Software used in the methodology for the analysis of SDS-PAGE gel electrophoresis (gel-electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate), is of paramount importance, since the degree of correlation between species depends on the parameters and algorithms used by this software. Without going into much detail, quantitative comparison of relationships, measured by the densitometer and normalized by the computer, preferably performed with the Pearson correlation coefficient. The matrix of similarity, thus obtained, can be formed using the algorithm UPGMA method (unweighted pair-wise clustering using average linkages), which not only allows you to group the most similar profiles, but also to create dendogram (see Kersters, Numerical methods in the classification and identification of bacteria by electrophoresis, in Computer-assisted Bacterial Systematics, 337-368, M.Goodfellow, A.G. O'donnell Ed., John Wiley and Sons Ltd, 1985).

Alternatively, the mentioned microorganisms belonging to the genus Lactobacillus may be assessed consider is Ino ribosomal RNA in the so-called Riboprinter.RTM. More preferably, the identity of the above species to the genus Lactobacillus is estimated by comparing the nucleotide sequence of the 16S ribosomal RNA of the indicated bacteria, or their genomic DNAs that encode the 16S ribosomal RNA, with those of other genera and species of lactic acid bacteria known to date. Another preferred alternative to determine the origin of species to the genus Lactobacillus is the use of species-specific PCR primers that targeted the spacer elements region 16S-23S p-RNA. Another preferred alternative is RAPD-PCR (PCR with random amplification of polymorphic DNA) (Nigatu et al. in Antonie van Leenwenhoek (79), 1-6, 2001), on the basis of this accumulating strain-specific set of DNA, which allows to determine the identity of the identifiable microorganisms of the genus Lactobacillus. Other techniques useful for detecting a microorganism belonging to the genus Lactobacillus are length polymorphism fragments (RFLP method) (Giraffa et al. Int. J. Food Environ. 82 (2003), 163-172), "fingerprinting repeating elements (Gevers et al., FEMS Environ. Lett. 205 (2001) 31-36) or the analysis of a set of methyl esters of fatty acids (fatty acid methyl ester, FAME) bacterial cells (Heyrman et al., FEMS Environ. Lett. 181 (1991), 55-62). Alternatively, the lactic acid bacteria can be determined by typing lectin (Annuk et al., . Med. Environ. 50 (2001), 1069-1074) or analysis of proteins in their cell walls (Gatti et al., Lett. Appl. Environ. 25 (1997), 345-348).

The above-mentioned microorganisms are preferably lactic acid bacteria belonging to genus Lactobacillus, more preferably of the Lactobacillus species, described here, in particular bacteria Lactobacillus belonging to a species selected from the group consisting of acidophilus, fermentum, lactis, delbrueckii, algidus, brevis, buchneri, casei, Camellia, coleohominis, crustorum, diolivorans, heterohiochii, hilgardii, kimchii, lindneri, oris, pantheris, parabuchner and saerimneri. Even more preferably the said Lactobacillus is Lactobacillus acidophilus. However, species of Lactobacillus are not limited to these. The above-mentioned microorganisms preferably can be "isolated" or "purified". The term "isolated" means that the material is withdrawn from the source environment, such as the natural environment if it is in a natural state. For example, isolated a naturally occurring microorganism, preferably of the Lactobacillus species isolated from some or all of the existing materials in the natural system. Such a microorganism can be part of a composition and still be considered isolated in it, if such a composition is not part of the natural environment.

The term "purified" does not require absolute purity; rather, it means relative definition wide-angle is. Individual microorganisms obtained from the library, were purified in the usual way to microbiological homogeneity, i.e. they grow as single colonies when seeded stroke on agar plates using methods known in the art. Preferably, agar cups that are used for this purpose, selective with respect to the species Lactobacillus. Such selective agar Cup known in the art.

More preferably, the above mentioned microorganism belonging to the group of lactic acid bacteria selected from the group consisting of bacteria Lactobacillus acidophilus, which has code DSM 19825, DSM 19826, DSM 19827 collection DSMZ, or mutants or derivatives, where the indicated mutant or derivative retains the ability to reduce the concentration of peptides in the analysis (a) and/or (A)described here above. The term "Lactobacillus acidophilus, having code in the collection DSMZ" refers to cells of a microorganism belonging to the species Lactobacillus acidophilus, deposited by the company BASF AG in cooperation with the German collection of microorganisms and cell cultures (Deutsche Sammlung fur Mikroorganismen und Zeilkulturen GmbH, DSMZ) on 1 November 2007 and codes with Deposit DSM 19825, DSM 19826, DSM 19827. Collection DSMZ is located on Inhoffenstr. 7b, 38124 Braunschweig, Germany.

The above DSMZ Deposit were made in accordance with the terms of the Budapest agreement on international the native recognition of the Deposit of microorganisms for purposes of patent procedure.

"Mutant or derivative" of the above mentioned microorganism belonging to the group of lactic acid bacteria, preferably deposited bacteria Lactobacillus acidophilus, preferably has the same characteristics that relate to the deposited strains, i.e. it retains the ability to reduce the concentration of peptides in the analysis (a) and/or (A)described herein above, and/or retains the ability to stimulate the growth of Streptococcus salivarius, but does not stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis. For example, this derivative can be created by means of genetic engineering. In the context of the present invention, the term "methods of genetic engineering" is used in its broadest sense to methods known to a person skilled in the technical field to modify the desired nucleic acid in vitro and in vivo so that genetic modification exposed to and genes were changed by recombinant DNA technology. Accordingly, it is preferable that these methods include cloning, sequencing and transformation of recombinant nucleic acids. For this purpose, suitable vectors, including expressing vectors for species of Lactobacillus, such as, for example, described in European patents EP-B1 506 789, EP-B1 316 677, EP-B1 251 064, EP-B1 218 230. EP-B1 133 046 or international patent application WO 89/01970.

The primers, the enzyme is, other cells-hosts for cloning intermediate structures and the like can be used and are known to the person skilled in the art. Preferably, the mutants, genetically engineered, contain cells of the above mentioned microorganism belonging to the group of lactic acid bacteria, preferably deposited species of Lactobacillus, including recombinant nucleic acid, or contained in the bacterial chromosome or plasmid(s). These recombinant nucleic acids alien preferably the above-mentioned microorganism belonging to the genus of lactic acid bacteria. Under "alien" means that the molecule polynucleotide or nucleic acid is either heterologous with respect to the cell master, that is obtained from a cell or organism with a different genomic environment, or is homologous to the cell master, but localized in a different genomic environment than the naturally occurring counterpart of the indicated molecules of nucleic acid. This means that if a molecule of nucleic acid is homologous to the cell master, it is not localized in its natural location in the genome of the specified host cell, in particular it is surrounded by other genes. In this case, polynucleotide m which may be either under the control of its own promoter, or under the control of a heterologous promoter. The above vector or nucleic acid molecule, which is present in the cell host, or may be integrated into the genome of the host cell, or may be supported in some form outside of the chromosome. In this regard, it should be understood that the above-described nucleic acid molecule can be used to restore or create a mutant gene via homologous recombination.

A mutant of the above mentioned microorganism belonging to the genus of lactic acid bacteria, preferably a mutant of the deposited strains of Lactobacillus, preferably is artificially mutated. According to the present invention, the term "mutant" refers to constant(s) modification(s) of the genetic material, i.e. nucleic acids, called(s), for example, naturally or by physical means or chemical compounds/substances/agents such as EMS (ethyl methanesulfonate) or ENMA (N-ethyl-N-nitrosamino). These modifications include point mutations, like transitions or transversum, deletions/insertions/attach one or more bases within the nucleic acid/gene/chromosome, thereby modifying the nucleic acid/gene/chromosome that can cause, among other things, aberrant gene exp is essay/transcription/translation inactive gene products, constitutive active/inactive gene products, leading, for example, a dominant negative effects. Preferably, the mutation leads to an increased ability to reduce the concentration of peptides in the analysis (a) and/or (A)described above. Thus, it is also preferable that in the mutant cells deposited microorganism that include the mutation(s) need(s) gene(s), this(and) mutation(s) need(s) gene(s) caused by methods known to the person skilled in the art. Also in the prior art it is known that the mutated or genetically engineered bacterial cells can be selected by any suitable method/phenotype. In the context of the present invention, the mutant with increased ability to reduce the concentration of peptides in the analysis (a) and/or (A)described above and/or having the ability to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis, can be tested in accordance with the methods described herein above or in the accompanying Examples. The term "mutant", however, also includes cells of the above mentioned microorganism belonging to the genus of lactic acid bacteria, preferably cells deposited microorganisms, which include what is happening in nature, a spontaneous mutation in the x genome, i.e. bacterial chromosome. Spontaneous mutations are mutations that occur in nature, i.e. without direct manipulation over the genetic material of human or exposure to a mutagen. Selection of spontaneous mutants can be done by kulturarbeit strain and selecting the desired options, for example, by the ability of various bacteria to show increased ability to reduce the concentration of peptides in the analysis (a) and/or (A)described above and/or having the ability to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis (see, for example, Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989)). For example, such mutations may occur during cultivation, for example, during the process of normal cell division, combined with DNA replication or during passage and/or storage mutant of the above mentioned microorganism belonging to the genus of lactic acid bacteria.

The present invention also relates to a derivative of the above mentioned microorganism belonging to the genus of lactic acid bacteria. The term "derivative of the above mentioned microorganism belonging to the genus of lactic acid bacteria" includes inactivated form, analog or fragment of Viseu manutoo microorganism, belonging to the genus of lactic acid bacteria, where specified inactivated form, analog or fragment retains the ability to reduce the concentration of peptides in the analysis (a) and/or (A)described above, and/or have the ability to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis.

According to the present invention, the term "inactivated form" includes a dead or inactivated cell according to the invention, which is no longer capable of forming a single colony on a Cup that is specific for microorganisms belonging to the genus Lactobacillus. The specified dead or inactivated cell can be either intact or damaged cell membrane. Methods of conciliation or inaktivirovanie cells of the above mentioned microorganism belonging to the genus of lactic acid bacteria, known in the art. Publication EI-Nezami et al., J. Food Prot. 61 (1998), 466-468 describes a method of inaktivirovanie species of Lactobacillus by UV-irradiation. Preferably, the cells of the microorganism according to the invention are thermally inactivated or liofilizirovannami, as described in the accompanying Examples. The lyophilization of cells, as described above, has the advantage that cells can easily be stored and they are easy to apply, while they retain their ability SN in order to reap the concentration of peptides in the analysis (a) and/or (A), described above, and/or have the ability to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis.

Moreover, lyophilized cells can grow again under conditions known in the art for the respective liquid or solid medium. The lyophilization is carried out by methods known in the art. Preferably, it is conducted for at least 2 hours at room temperature, i.e. any temperature between 16°C and 25°C. is Particularly preferable to carry it out for 16 hours in vacuum. Moreover, freeze-dried cells of the above mentioned microorganism belonging to the genus of lactic acid bacteria, is stable for at least 4 weeks at 4°C, so that they retain the ability to reduce the concentration of peptides in the analysis (a) and/or (A)described above, and/or the ability to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis.

Thermal inactivation can be achieved by incubation of the cells of the above mentioned microorganism belonging to the group of lactic acid bacteria, at least for 10 minutes at a temperature of 80°C. Thermal inactivation can be carried out by autoclaving these cells and/or supernatant at a temperature of 121°C for at less the th least 20 minutes in the presence of saturated steam at atmospheric pressure 2 bar. Preferably, thermal inactivation of the cells or culture supernatant is carried out as described here above with reference thermal processing.

Alternatively, thermal inactivation of the cells of the above mentioned microorganism belonging to the group of lactic acid bacteria by freezing these cells for 4 weeks, 3 weeks, 2 weeks, 1 week, 12 hours, 6 hours, 2 hours or 1 hour at -20°C. it is Preferable that at least 70%, 75% or 80%, more preferably 85%, 90% or 95% and especially preferably at least 97%, 98%, 99% and more preferably 99,1%, 99,2%, 99,3%, 99,4%, 99,5%, 99,6%, 99,7%, 99,8% or 99.9% and most preferably 100% of the cells of the analogue of the above mentioned microorganism belonging to the group of lactic acid bacteria, dead or inactivated, however they still have the ability to reduce the concentration of peptides in the analysis (a) and/or analysis (A)described herein above, and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis. Are there really inactivated form, analog or fragment of the above mentioned microorganism belonging to the group of lactic acid bacteria, dead or inactivated, can be determined using methods known in the art, for example using the test of life is sposobnosti.

The term "inactivated form" or "analog" also includes lysates, fractions, such as membrane fractions or extracts of the above-described microorganisms, where these lysates, fractions or extracts retain the ability to reduce the concentration of peptides in the analysis (a) and/or analysis (A)described herein above, and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis. These abilities can be tested as described herein and, in particular, as described in the accompanying Examples. If the lysate, fraction or extract of the microorganism according to the invention described here above may not show the ability to reduce the concentration of peptides in the analysis (a) and/or analysis (A)described herein above, and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis, then the specialist in the art may, for example, further purify these lysate, fraction or extract using methods known in the art, as illustrated by the examples here below, in order to remove substances which inhibit the reduction. After that, the specialist in the art can again test the specified lysate, fraction or extract, is he able to reduce the concentration of peptides in the analysis (a) and/or (A)described here above.

According to the SNO present invention, the term "lysate" means a solution or suspension in an aqueous medium of the cells of the microorganism according to the invention. However, the term should not be interpreted in any limiting manner. The cell lysate contains, for example, macromolecules such as DNA, RNA, proteins, peptides, carbohydrates, lipids and the like, and/or macromolecule, such as amino acids, sugars, lipid acid and the like, or their fractions. Additionally, the specified lysate contains the cellular detritus, which may have smooth or granular structure. Preferably, the specified lysate contains the cell wall or cell membrane, or both, or parts or fragments of the cell wall or cell membrane, or both. Methods for obtaining cell lysate of a microorganism known in the art, for example, the use of the French, cellular mill using glass or iron balls, or enzymatic cell lysis, and the like. Additionally, the lysis of cells refers to various methods known in the art for opening/destruction of cells. Method of lysis of the cells is not important and you can use any method by which it is possible to achieve cell lysis, the above mentioned microorganism belonging to the genus of lactic acid bacteria. The appropriate method can be selected by a person skilled in the technical field, for example the opening/destruction of cells can be accomplished enzymatically, chemically or physically. Neagra Cialdini examples of enzymes and enzyme cocktails are protease, as protease, lipase or glycosidase; non-limiting examples of chemical substances are ionophores, detergents like sodium dodecyl sulphate, acid or base; and non-limiting examples of physical means are high pressure, as French press, osmolarity, temperature, as heat or cold. For example, cells of the above mentioned microorganism belonging to the genus of lactic acid bacteria are lysed by freezing and thawing, more preferably freeze at temperatures below -70°C and melting at temperatures greater than 30°C, especially freezing preferably at temperatures below -75°C and the melt, preferably at temperatures higher than 35°C and most preferably at temperatures below freezing to -80°C and melting temperatures over 37°C. Also preferably, the specified freezing/thawing was repeated at least 1-fold, more preferably at least 2 times, more preferably at least 3 times, particularly preferably at least 4 times and most preferably at least 5 times.

Accordingly, specialists in the art can achieve the desired lysates, referring to the above General explanation and appropriately modifying or altering these methods if necessary. Preferably, water is the medium, used to described lysates is water, physiological saline or buffer solution. The advantage of bacterial cell lysate is that it can be easily obtained and effectively to save costs, as it requires less technical base.

Preferably, the term "extract" means a subcellular component of the above mentioned microorganism belonging to the genus of lactic acid bacteria, such as a macromolecule, such as protein, DNA, RNA, peptide, carbohydrate, lipid and the like, and/or macromolecule as amino acid, sugar, lipid acid and the like, or any other organic compound or molecule, or a combination of these macromolecules, and/or macromolecule or any fraction where the specified extract retains the ability to reduce the concentration of peptides in the analysis (a) and/or (A)described above, and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis. These properties can be tested as described herein or, in particular, as described in the accompanying Examples. Preferably, this extract contains the cell wall or cell membrane, or both, or parts or fragments of the cell wall or cell membrane, or both. More preferably, the term "extract" refers to any of the above you is E. subcellular component in a cell-free environment.

In another preferred embodiment of the invention, the extract can be obtained by lysis of cells in accordance with various methods known in the art for opening/destruction of cells, as described above, and/or may be a supernatant procedures centrifugation of the culture of the above mentioned microorganism belonging to the genus of lactic acid bacteria, in any suitable liquid medium or buffer known to the person skilled in the technical field, or a lysate of such a culture, or any other suitable cell suspension. More preferably, the extract can be purified extract or cell culture supernatant, or any faction or subcaste where indicated cleared lysate or cell culture supernatant, or any faction or subcaste retains the ability to reduce the concentration of peptides in the analysis (a) and/or (A)described above and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis. These properties can be tested as described above and, in particular, as described in the accompanying Examples. Suitable methods for fractionation and purification of the lysate, culture supernatant or extract well-known specialist in the art and include, for example, affinity chromatography, and noobman chromatography, exclusion chromatography, reversed-phase chromatography and chromatography with other chromatographic material in the column or methods of group processing, other methods of fractionation, for example, filtering methods, such as ultrafiltration, dialysis, dialysis and concentration with an exclusion size by centrifugation, centrifugation in density gradient or step-by-step matrix deposition, for example, affinity precipitation, precipitation with salivarium or by salting out (precipitation with ammonium sulfate), alcohol precipitation, or any other protein-chemical, molecular, biological, biochemical, immunological, chemical or physical method.

According to the invention lysates are also preparations or fractions of molecules of the above lysates. These fractions can be obtained by methods known to the person skilled in the art such as chromatography, including affinity chromatography, ion exchange chromatography, exclusion chromatography, reversed-phase chromatography, and chromatography with other chromatographic material in the column or methods of group processing, other methods of fractionation, for example, filtering methods, such as ultrafiltration, dialysis, dialysis and concentration with an exclusion size by centrifugation, centrifuger the tion in the density gradient or step-by-step matrices, deposition, for example, affinity precipitation, precipitation with salivarium or by salting out (precipitation with ammonium sulfate), alcohol precipitation, or any other protein-chemical, molecular, biological, biochemical, immunological, chemical or physical methods to separate the above component lysates. In a preferred embodiment of the invention preferred are those fractions which are more immunogenic than others. Specialists in the art can select a suitable method and to determine the immunogenic potential, referring to the above General explanation and specific explanations in the examples and appropriately modifying or altering these methods if necessary.

Accordingly, the term "inactivated form of analog" also covers the filtrates of the microorganism of the invention, where these filtrates preferably retain the ability to reduce the concentration of peptides in the analysis (a) and/or (A)described above and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis. These properties can be tested as described herein and, in particular, as described in the accompanying examples. When the filtrate of the above mentioned microorganism belonging to the genus Molochnik slyh bacteria, as described above, may not be able to reduce the concentration of peptides in the analysis (a) and/or (A)described above and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis, then the person skilled in the art may, for example, continue to clear the specified filtrate techniques known in the art and illustrated by the examples here below, to remove substances that inhibit the reduction and/or growth stimulation. After that, the specialist in the art can again be specified test the filtrate for the ability to reduce the concentration of peptides in the analysis (a) and/or (A)described above and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis.

The term "leachate" means a cell-free solution or suspension of the microorganism of the invention described here above, which were obtained as supernatant procedures centrifugation of the culture of the above mentioned microorganism belonging to the genus of lactic acid bacteria, in any suitable liquid medium or buffer known to the person skilled in the art. However, the term should not be interpreted in any limiting manner. The filtrate contains, for example, macromolecules such as DNA, RNA, proteins, peptides, carbohydrates, lipids and the like, and/or micromoles is s, as amino acids, sugars, lipid acid and the like, or their fractions. Methods of obtaining filtrates of microorganisms known in the art. Additionally, the "filtrate" refers to various methods known in the art. The selected method is not important and you can use any method by which you can reach the filter cells of the microorganism of the invention. The term "filtrate" also includes cultural supernatant, for example, obtained by sedimentation of the cells by centrifugation and recovery of the resulting supernatant.

In a particularly preferred embodiment of the invention the filtrate, more preferably, the culture supernatant is processed in a different way, in particular thermally or by lyophilization, as described here above.

"Fragment" of a microorganism according to the invention covers any portion of the cells of the above mentioned microorganism belonging to the genus of lactic acid bacteria. Preferably, the specified portion is a membrane fraction obtained through the preparation of membranes. Membrane preparations of microorganisms belonging to the genus Lactobacillus, can be obtained by methods known in the art, for example using the method described in publications Rollan et al. Int. J. Food Environ. 70 (2001), 303-307, Matsuquchi et al. Clin. Dagn. Lab. Immunol. 10 (2003), 259-266 or Stentz et al. Appl. Environ. Environ. 66 (2000), 4272-4278 orVarmanen et al. J. Bacteriology 182 (2000), 146-154. Alternatively, the preparation of whole cells is also provided. Preferably, there is described a derivative or fragment of the above mentioned microorganism belonging to the genus of lactic acid bacteria, retains the ability to reduce the concentration of peptides in the analysis (a) and/or (A)described above and/or to stimulate the growth of Streptococcus salivarius and not to stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis.

The present invention also relates to compositions containing the above-mentioned microorganism according to the present invention, its inactivated form, derivative, or mutant, or an analogue or fragment. The composition is preferably a cosmetic composition, or pharmaceutical composition, for example for the treatment and/or prevention of unpleasant mouth odor and/or halitosis, or feed, or food composition.

In a preferred embodiment of the invention, the composition contains the above-described microorganism in amounts of between 102up to 1012cells, preferably from 103up to 108cells, mg in the solid form of the composition. In the case of compositions in liquid form, the number of microorganisms is between 102up to 1013cells / ml, However, Conques is to maintain the compositions of the amount of the microorganism may vary, as described here.

The cosmetic composition may contain a carrier or excipient that is acceptable for cosmetic or oral use. The pharmaceutical composition may contain a carrier or excipient that is acceptable for pharmaceutical or oral use. Feed or food composition may contain a carrier or excipient that is acceptable for oral use.

The present invention also relates to the use of a microorganism according to the present invention, its inactivated form, derivative, or mutant, or analog, or fragment for the preparation of compositions containing the above-mentioned microorganism according to the present invention, its inactivated form, derivative, or mutant, or similar, or a fragment, especially a cosmetic composition, feed or food composition or pharmaceutical composition for prevention and/or treatment of bad breath and/or halitosis.

Such a composition can be prepared by a method containing the step of compiling the microorganism according to the invention with a carrier or excipient that is acceptable for cosmetic, oral or pharmaceutical use.

The term "composition", as used according to the present invention, relates to the composition(s), to the which(s) contains(at) at least one microorganism, or a mutant or derivative described above, or inactivated form, or similar, or a fragment of a specified microorganism. It is envisaged that the compositions used according to the present invention, which are described here below, contain the above components in any combination. They may optionally contain at least one other component suitable for the prevention and/or treatment of bad breath and/or halitosis. Accordingly, they can contain any combination of the above on the other ingredients. The term "components suitable for the prevention and/or treatment of bad breath and/or halitosis" includes compounds or compositions and/or combinations thereof that are known in the art to reduce unpleasant mouth odor. Examples are metal salts such as zinc chloride, disinfectants such as alcohol or chlorhexidine, which is used for treatment of language. Another example are compounds that help to maintain the pH of saliva to a physiologically normal level. It can be substances with pH-raising or pH-buffering agents (e.g., bicarbonate, urea, phosphates, proteins and/or salt). It is known that species of microbes associated with caries and infections clinicolaboratory, favorable to the development of an acidic environment, types of microbes associated with the development of periodontal disease, favor the development of pH above normal, while the varieties of microbes associated with good health of your mouth, favor the development of neutral pH. Another example is the probiotic bacteria (such as Lactobacillus and Streptococcus)disclosed in patent applications U.S. US 200707137, US 2006018843 or international patent application WO 2007/077210, or lactic acid bacteria belonging to the genus Weisella, which inhibit the growth of LSS-producing bacteria by interacting with them and generating hydrogen peroxide under aerobic and anaerobic conditions, as described in application for U.S. patent US 2006045870. Finally, BPIV (bacteriocins-like inhibiting substances) - producing strains of Streptococcus salivarius and their extracts can be mentioned in this context (application for U.S. patent US 2006171901).

Indicates that the composition used in accordance with the present invention can optionally contain one or more of the above optional components, which are suitable for the prevention and/or treatment of bad breath and/or halitosis. Thus, this composition may contain at least two, three, four, five, etc., i.e. the "n" optional components, where "n" is the tsya integer, greater than 2 and without restrictions. These optional components can be combined in any possible combination.

The composition may be in solid, liquid or gaseous form and may be, inter alia, in powder form(s)tablet(s), film(s) of drug(s)solution(s)aerosol(s), granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures or liquid extracts, or in a form that is particularly suitable for oral administration.

Liquid preparations suitable for oral administration such as syrups, can be prepared using water, normal sugars such as sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, antiseptics such as an ester of n-hydroxybenzoate, preservatives, such as derivatives of n-hydroxybenzoate, for example n-hydroxybenzoate methyl and sodium benzoate, and other materials, such as flavorings, such as strawberry flavor or peppermint.

Further preparations suitable for oral administration such as tablets, powders and granules can be obtained using conventional sugars such as sucrose, glucose, mannitol and sorbitol, starch, such as potatoes, wheat and grain, inorganic materials, is such as calcium carbonate, calcium sulfate, sodium bicarbonate and sodium chloride, powder from plants, such as crystalline cellulose, powder, licorice powder and gentian, fillers such as pinedex, disintegrators such as starch, agar, gelatin powder, crystalline cellulose, carmellose sodium, carmellose calcium, calcium carbonate, sodium bicarbonate and sodium alginate, lubricants such as magnesium stearate, talc, hydrogenated vegetable oil, macrogol or silicone oil, binder, such as polyvinyl alcohol, hydroxypropylcellulose, methylcellulose, ethylcellulose, carmellose, gelatin solution, starch paste, surfactants, such as an ester of a fatty acid, and a plasticizer, such as glycerol. Film(s) drug(s) may be prepared(s) by methods known in the art. Example of preparation of a film is given in Example 19.

In the case of the usual oral dose of the above-described microorganism, or analog, or fragment may be in dry weight as described above in relation to the number of cells or in relation to the mass, for example, from 1 mg to 50 g, from 1 μg to 10 g, from 1 μg to 5 mg, 1 μg to 1 mg or any other dose to a subject in a day or in several portions a day. Further, in the case of dosing animals dose varies over the threaded depending on the age and species of animal and nature or the severity of symptoms. Without limiting the dose for animals ranging from 0.1 mg to 10 g per 1 kg of body weight, preferably from 1 mg to 1 g per 1 kg of body weight once or in several portions a day. However, these dose and number of doses vary depending on individual conditions.

Preferably, the composition in accordance with the present invention is a cosmetic composition, optionally containing a cosmetically acceptable carrier or excipient. More preferably, the composition is a means for brushing your teeth, chewing gum, tablet, mouthrinse, tooth elixir, dental floss or dental tape.

Cosmetic composition in accordance with the present invention contains the microorganism, its inactivated form, mutant, derivative, analog or fragment, as described above in connection with the composition of the invention, and a carrier acceptable for cosmetic or oral use. Preferably, as noted in connection with the composition in accordance with the present invention, the microorganism, inactivated form, mutant, derivative, analog or fragment represent the microorganism, its inactivated form, mutant, derivative, analog or fragment described herein above. Preferably, the cosmetic to notice according to the present invention is intended for use in the oral cavity. Accordingly, it may be in the form of toothpaste, cleaning teeth, tooth powder, gel for topical application in the oral cavity, dental elixir, means for dentures, aerosol for flavoring mouth, sucking tablets, oral tablets, chewing gum, dental floss or dental tape.

The term "carrier acceptable for oral or cosmetic use"used here means a suitable carrier medium, which may be used to apply the present compositions to the oral cavity a safe and effective manner. Such a carrier medium may include materials, such as sources of fluoride ions, additional anticellulite agents, buffers, and other abrasive materials, sources of hydrogen peroxide, bicarbonate alkali metal salts, thickeners, humectants, water, surfactants, titanium dioxide, flavorings, sweeteners, xylitol, coloring agents and mixtures thereof. The term "safe and effective amount", as used here, means a quantity sufficient to clean teeth and reduce pigmentation/plaque/gingivitis/dental stones without damage to the tissues and structures of the oral cavity.

The pH of the compositions described herein is preferably in the range from about 3.0 to about 9.0, with predpochtitel the hydrated pH is from about 5.5 to about 9.0, and most preferred pH is from 7.0 to about 8.5 or 9.0.

Cosmetic composition is a tool, which in normal use will not swallow intentionally for the purpose of system the introduction of particular therapeutic agents, but rather keep him in the oral cavity for a time sufficient to contact almost all of the dental surfaces and/or oral tissues, with the target of the action in the oral cavity. The oral composition may be single-phase oral composition or may be a combination of two or more compositions to the oral cavity.

The term "means for cleaning the teeth"used here means a paste, gel or liquid compositions, unless otherwise specified. Composition for cleaning teeth can be in any desired form, such as with deep stripes, with surface bars, multilayer, having a gel around pasta or combinations thereof. Composition for cleaning teeth may be in physically separate chamber of the dispenser or placed closely. Composition means for brushing disclosed, for example, in European patent EP-B1 0617608.

Preferred compositions of the means for cleaning the teeth disclosed in Examples 13-16. In addition to the above-described components of the composition means for cleaning the teeth of this invention may contain other optional components for brushing your teeth, some the activities of which are described below. Optional components include, but are not limited to, for example, connecting components, foaming agents, flavorings, sweeteners, additional agents against dental plaque, additional abrasive materials and dyes. These and other desired components are also described, for example, in U.S. patents US 5,004,597; US 4,885,155; US 3,959,458; and US 3,937,807.

For example, toothpaste may include surfactants, chelating agents, sources of fluoride, the active substance for whitening teeth and substances that alter the color of teeth, thickeners, humectants, flavors and sweeteners, bicarbonate salt of an alkali metal, mixed media, and/or other active agents.

One of the preferred optional agents to be used in accordance with the present invention is a surfactant, preferably a surface-active substance (surfactant)selected from the group consisting of sarcosinate surfactants, isethionate surfactants and taurate surfactant. Preferred for use are alkali metal salts and ammonium of these surfactants. Most preferred are the sodium and potassium salts of the following surfactants: lauroyl sarcosinate, myristoyl sarcosinate, Palmitoyl sarcosinate, stearoyl sarcosinate and oleoyl sarcosinate.

Other preferred optional agent is m is a chelating agent, such as tartaric acid and its pharmaceutically acceptable salts, citric acid and alkali metal citrates and mixtures thereof. Chelating agents capable of forming a complex with the calcium found in the cell walls of bacteria. Chelating agents may also disrupt the plaque due to the removal of calcium from calcium bridges that help to keep the biomass intact.

To ensure the effectiveness against caries is considered to have an additional water-soluble fluoride compound, which is present in the means for brushing your teeth and other compositions for the oral cavity in a quantity sufficient to create a concentration of fluoride ions in the composition at 25°C., and/or at a concentration of from about 0,0025 to about 5.0 wt.%, preferably from about 0,005 to about 2.0 wt.%. A wide variety of materials, giving the fluoride ions can be used as sources of soluble fluoride in these compositions. Examples of suitable materials that provide fluoride ions found in the U.S. patents US 3,535,421 and US 3,678,154. Representatives of the sources of fluoride ions include tin fluoride, sodium fluoride, potassium fluoride, monitoroff sodium and many others. The tin fluoride and sodium fluoride are particularly preferred, as well as mixtures thereof.

Compositions for the care of the oral cavity of the present invention can also contain active the e substance for whitening teeth including bleaching or oxidizing agents such as peroxide, perborate, percarbonate, peroxy acids, persulfates, chlorites metals, and combinations thereof. Suitable connections peroxides include hydrogen peroxide, urea peroxide, calcium peroxide, and mixtures thereof. The preferred percarbonate is percarbonate sodium. Other suitable bleaching agents include potassium, ammonium, sodium and lithium persulfates and perborate mono - and tetrahydrate, and sodium pyrophosphate peroksigidrat. Suitable metal chlorites include chlorite calcium, barium chlorite, magnesium chlorite, lithium chlorite, sodium chlorite and potassium chlorite. The preferred chlorite is sodium chlorite. Additional bleaching active substances can be hypochlorite and chlorine dioxide.

In addition to the bleaching agents, such as agents for whitening teeth substances, changing the color of the teeth, can be considered among the active substances for the care of the oral cavity useful in the present invention. These substances are suitable for changing the color of teeth, to satisfy the wishes of the consumer. These substances contain particles, which when applied to the tooth surface to modify the surface from the point of view of absorption and/or reflection of light. Such particles have a beneficial effect on the appearance when using film, maderaspatana particles, on the surface of the tooth or teeth.

In the preparation of toothpastes or gels it is necessary to add some thickening agent to provide the composition the desired consistency, to provide the desired release characteristics of the active substance in use, to ensure the storage stability and the stability of the composition, etc. Preferred thickeners are carboxyvinyl polymers, carrageenan, hydroxyethyl cellulose, LAPONITE®(manufactured by Rockwood, Additives limited) and water-soluble salts of cellulose ethers such as carboxymethylcellulose sodium and karboksimetiltselljuloza sodium. You can also use natural gums such as gum karaya, xanthan gum, Arabic gum and tragacanth gum. Colloidal magnesium silicate aluminum or highly dispersed silicon dioxide can be used as part of the thickening agent to improve the texture.

Another optional component carriers for local oral administration of the compositions of the claimed invention is a humidifier. The humectant serves to protect the compositions of toothpastes from hardening on exposure to air, gives the composition in the mouth the feeling of humidity and, for certain humidifiers, gives the desired sweet taste to the composition of toothpastes. Umlani the spruce, in the calculation of the net humidifier, usually contains from about 0% to about 70%, preferably from about 5% to about 25 wt.% from the composition. Suitable humectants for use in compositions of the claimed invention include edible polyhydric alcohols such as glycerin, sorbitol, xylitol, butylene glycol, polyethylene glycol and propylene glycol, especially sorbitol and glycerin.

Flavors and sweeteners can also be added to the compositions. Suitable flavoring agents include oil of Grushenka, oil of peppermint, oil of spearmint, oil of flowers, clove, menthol, anethole, methyl salicylate, eucalyptol, Cassia, 1-Menthyl acetate, sage, eugenol, oil of parsley, Oksanen, alpha ireson, marjoram, lemon, orange, propergate, cinnamon, vanillin, thymol, linalool, glycerin acetal cinnamic aldehyde, known as CGA, and mixtures thereof. Flavoring agents are generally used in the compositions at levels from about 0,001% to about 5 wt.% from the composition.

Sweeteners that can be used include sucrose, glucose, saccharin, dextrose, levulose, lactose, as described herein above, lures, sorbitol, fructose, maltose, xylitol, salt saccharin, thaumatin, aspartame, D-tryptophan, dihydrochalcones, salts of Acesulfame and cyclamate, especially sodium cyclamate and saccharin sodium, and mixtures thereof. The composition preferably contains from okolo,1% to about 10% of these agents, preferably from about 0.1% to about 1 wt.% from the composition.

The composition of the present invention may also include bicarbonate salt of an alkali metal. Bicarbonate salts of alkali metals are soluble in water and, if not stable, tend to release carbon dioxide in an aqueous system. Sodium bicarbonate also known as baking soda, is the preferred alkali metal salt. The present composition may contain from about 0.5% to about 30%, preferably from about 0.5% to about 15%, and most preferably from about 0.5% to about 5% of the bicarbonate salt of an alkali metal.

Water used in the preparation suitable for industrial environments compositions should preferably be of low ion content and free of organic impurities. Water usually contains from about 10% to about 50%, and preferably from about 20% to about 40%, by weight of the aqueous compositions of toothpastes. These amounts of water include the free water which is added plus that which is introduced with other materials, such as sorbitol. Titanium dioxide may also be added to the present composition. Titanium dioxide is a white powder that adds the turbidity of the compositions. Titanium dioxide typically contains from about 0.25% to about 5% by weight of the compositions for cleaning teeth.

The pH of the compositions of the image is preferably placed regulate through the use of buffering agents. Buffer agents used herein, refer to agents that can be used to adjust the pH of the compositions at a level of from about 4.5 to about 9.5. Buffer agents include phosphate monolatry, trisodium phosphate, sodium hydroxide, sodium carbonate, sodium pyrophosphate, citric acid and sodium citrate. Buffer agents may be introduced at the level of from about 0.5% to about 10 wt.% from these compositions. The pH of the compositions for cleaning teeth is measured for a ratio of 3:1 water suspension means for brushing your teeth, for example 3 parts water to 1 part toothpaste.

Other optional agents that can be used in these compositions include Dimethicone copolyol selected from alkyl and alkoxylation of copolyol, such as C12-C20 alkyldimethyl copolyol and mixtures thereof. Highly preferred is acidisation copolyol sold under the trade name Abil EM90. Dimethicone of copolyol typically present at a level from about 0.01% to about 25%, preferably from about 0.1% to about 5%, more preferably from about 0.5% to about 1.5 wt.%. Dimethicone copolyol help ensure a beneficial effect on the teeth. Other useful carriers include bi-phase formulation of the means for cleaning the teeth, such as those disclosed in U.S. patents US 5.213.790; US 5.145.666; US 5.281.410; US 4.849.213 and US 4.528.180.

The cosmetic composition may also include other active agents, such as antimicrobial agents. Such agents include water-insoluble nicotianae antimicrobial agents such as halogenated diphenyl ethers, phenolic compounds including phenol and its homologs, mono - and polyalkyl and aromatic halophenol, resorcinol and its derivatives, bisphenol compounds and halogenated salicylanilides, benzoic esters, and halogenated carbanilide. Water-soluble antimicrobial agents, among others, include salts of Quaternary ammonium, and salts of bis-biguanide. Triclosan monophosphate is more water-soluble antimicrobial agent. The Quaternary ammonium compounds include those in which one or two of the substituents on the Quaternary azoth have a carbon chain length (typically alkyl group) from about 8 to about 20, typically from about 10 to about 18 carbon atoms while the remaining substitutes (typically alkyl or benzyl group) have a lower number of carbon atoms, such as from about 1 to about 7 carbon atoms, typically a methyl or ethyl group. Dodecyltrimethylammonium bromide, tetradecylbenzene chloride, domiphen bromide, N-tetradecyl-4-ethylpyridine chloride, dodecyldimethyl(2-phenoxyethyl)ammonium bromide, benzyldimethyl the Rhyl ammonium chloride, cetylpyridinium chloride, and stereoselectivity 5-amino-1,3-bis(2-ethylhexyl)-5-methylhexahydrophthalic, benzalkonium chloride, benzene chloride and methylbenzethonium chloride are examples of typical antimicrobial agents of Quaternary ammonium. Other compounds are bis[4-(R-amino)-1-pyridinium] alkanes disclosed in U.S. patent US 4,206,215. Other antimicrobial agents such as bisglycinate copper, glycinate copper, zinc citrate and zinc lactate, may also be included. Enzymes are another type of active substances, which can be used in these compositions. Useful enzymes include those that belong to the category of proteases, lytic enzymes, inhibitors of the matrix of dental plaque and oxidase. Proteases include papain, pepsin, trypsin, fitsin, bromelain; lytic enzymes cell wall include lysozyme; inhibitors of the matrix of dental plaque include dextranase, atanazy; and oxidases include glucose oxidase, lactate oxidase, galactose oxidase, oxidase uric acid, peroxidase, including horseradish peroxidase, myeloperoxidase, lactoperoxidase, chloroperoxidase. In addition to antimicrobial properties oxidase also have whitening/cleaning activity. Such means are disclosed in U.S. patents US 2.946.725 and US 4.051.234. Other antimicrobial agents include PI is lexigen, triclosan monophosphate and aromatic oils, such as thymol. Triclosan and other agents of this type are disclosed in U.S. patents US 5.015.466 and US 4.894.220. These agents, which are effective against dental plaque may be present at levels from about 0.01% to about 5.0 wt.% from the composition for cleaning teeth.

The term "chewing gum", as defined here, means confectionery composition, which is suitable for chewing and which contains any suitable number of elastomer known to the person skilled in the technical field, preferably the amount is 2% or more by weight of the composition. Suitable components for tablets for sucking and chewing gum are disclosed, for example, in U.S. patents US 4.083.955; US 6.770.264 and US 6.270.781. Preferred tablets for sucking are those that are disclosed in Examples 11 and 12. The preferred chewing gum composition disclosed in Example 17.

Compositions according to the present invention preferably contain an elastomer or a mixture of several different elastomers. Elastomeric materials commonly known in the art, illustrative examples include the best choice rubber (SBR); synthetic resins; polyisobutylene and isobutylene-isoprene copolymers; natural resins; cicli; natural rubber; jelutong, Balata; gutta-percha; Lechi Caspi; is the ODS and their mixtures. Compositions of the present invention preferably contain from about 2% to about 30%, more preferably from about 5% to about 25%, by weight of the elastomer. These levels are determined taking into account the desired final texture of chewing gum, because, when the total level of elastomer below about 2%, the base composition does not have enough elasticity, chewy texture and the ability to adhesion, while levels above 30% make the structure solid, rubbery and provide heavy chewing. Solvents elastomers are also preferably present in the compositions of the present invention, as they help to soften the elastomeric component. Preferred examples of elastomer solvents for use here include pentaerythritol ester partially gidrirovannoe extraction of rosin, pentaerythritol ester extraction of rosin, glycerol ester of partially dimenisonal rosin, glycerol ester of polymerized rosin, glycerol ester of tall oil, extraction or gum rosin, glycerol ester of partially gidrirovannoe rosin, methyl ester of partially gidrirovannoe rosin and mixtures thereof. Compositions of the present invention preferably contain from about 2 wt.% to about 50 wt.%, more predpochtitel is about from about 10 wt.% to about 35 wt.% solvent elastomers.

Lozenges according to the present invention can be prepared, for example, using accepted in the art methods for forming compressed tablets, where the disaccharide dispersed in the compression of the solid carrier, optionally combined with any suitable tabletting AIDS such as a lubricating substance (for example, magnesium stearate, and compressed into tablets. Solid component carrier for such compositions for tabletting may be soluble in saliva solid substance, such as a dry water-soluble starch or a monosaccharide to the ODT tablet easily dissolved when its holding in the mouth, releasing the contained disaccharide glycosides of the acid in the saliva solution for contact and absorption by the mucous membrane of the mouth or faringealni mucosa. The pH of the above-described compositions may be in the range of from about 4 to about 8.5.

Lozenges according to the present invention can also be prepared using other methods approved in the art for preparing solid compositions with a single dose.

A mouthrinse or mouthwash according to the present invention preferably may be as follows:

And Oil Menthae1.2 part
Tincture Arnicaeto 3.0 parts
Tincture Myrrhaeto 3.0 parts
Tween5.0 parts
B Alcohol 90%50.0 parts
In sodium Benzoate0.2 parts
Sweetener (for example,
part of aspartame)0,02
Distilled water100

And should be well mixed, with stirring, is added to B and then added C. the resulting clear liquid must be filtered within 48 hours after preparation. Another preferred mouthrinse described in Example 18.

Regardless of the dosage form, liquid or solid, in one preferred embodiment of the present invention, the dosage form is kept in the patient's mouth during the period, contributing to a contact of the microorganism, or analog or fragment of the above mentioned microorganism belonging to the genus Molochnik slyh bacteria, with oral cavity of the patient.

The terms "dental floss and dental tape"used here refer to the material for the abatement and removal of decaying food debris that accumulate in the interdental and subgingival intervals and to displace and remove bacteria, plaque and/or dental stones that accumulate in the mouth. Dental floss or dental tape in addition to the microorganisms according to the present invention, described above, may also contain cleaners, abrasives, controlling plaque components, bleaches, surfactants and/or active ingredients, such as fluoride, antimicrobial agents, chemotherapeutic agents, or antibiotics. Other additional methods are anti-plaque, flavorings and colorings. Dental floss or dental tape can be in any suitable form known to the person skilled in the technical field, for example in the form of dental floss, PTFE (Teflon), are described, for example, in U.S. patents US 3.664.915, US 3,953,566, US 3,962,153, US 4,096,227. US 4,187,390, US 4,256,806. US 4,385,093, US 4,478,665, US 4,776,358. US 5,033,488. US 5,209,251. US 5,220,932, US 5,518,012, US 5.718.251, US 5,765,576 or US 5,911,228, in the form of interdental devices from monofilament described, for example, in U.S. patents US 3,800,812, US 4,974,615, US 5,760,117, US 5,433,226, US 5,479,952, US 5,503,842. US 5,755,243, US 5,884,639, US 6,003,525 or US 6,027,592, or in the form biocomponent tapes. Preference is sustained fashion, dental floss or dental tape can be in the form of the elastomer coated monofilament described, for example, in the application for U.S. patent US 20050226820, or in the form of dental tape on the basis of oriented thermoplastic material is described, for example, in the application for U.S. patent US 20020144704.

Cosmetic compositions described herein above, can be used for oral administration to man, as well as oral administration to an animal, preferably a mammal, not belonging to the human race, more preferably pet. If the cosmetic composition is used for oral administration to an animal, the composition may contain other components such introduction, the well-known specialist in this field of technology.

The present invention also relates to the use of a microorganism according to the invention or its inactivated form for the preparation of pharmaceutical compositions for the treatment and/or prevention of bad breath and/or halitosis. Preferably, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

The pharmaceutical compositions contain a therapeutically effective amount of the microorganism of the present invention and can be prepared in different forms, such as solid, liquid, powder, water, lyophilize the consistent form.

The pharmaceutical composition can be entered with a pharmaceutically acceptable carrier to the patient, as described herein. In a specific embodiment of the invention, the term "pharmaceutically acceptable" means approved by a regulatory body or other widely recognized Pharmacopoeia for use of animals and, in particular, to the people.

The term "carrier" refers to a solvent, auxiliary substance, excipient or carrier environment, which introduces a therapeutic tool. This carrier is pharmaceutically acceptable, i.e. non-toxic with respect to the recipient in the applied dosage and concentration. Preferably it is isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, such as network sucrose solution. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous dextrose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, sucrose, gelatin, malt, rice, powder is to, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ion, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like, the Filler may contain lactose, as described above, more preferably lactose is absent. The composition, if desired, can also contain minor amounts of wetting agents or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, compositions with delayed release and other Oral formulation can include standard carriers such as pharmaceutical brand mannitol, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc. are Examples of suitable pharmaceutical carriers are disclosed in the publication "Remington''s Pharmaceutical Sciences" by E.W.Martin. Skimmed milk, skimmed milk powder, products that do not contain milk or lactose, can also be used. Skimmed milk is traditionally suspended in phosphate-buffered saline (FSB), autoclave or filter to remove protein or alive impurities, and then dried by sublimation, thermally dried, dried in vacuum or lyophilizer. Some other examples of substances which can serve as pharmaceutical carriers are sugars such as glucose and sugar is a; starches, such as corn starch and potato starch; cellulose and its derivatives, such as carboxymethylcellulose sodium, ethyl cellulose acetates and cellulose; powdered tragakant; malt; gelatin; talc; stearic acid; magnesium stearate; calcium sulfate; calcium carbonate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of Theobroma; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; agar; alginic acid; pyrogen-free water; isotonic saline; cranberry extracts and phosphate buffer solution; skimmed milk powder; as well as other non-toxic compatible substances used in pharmaceutical formulations such as vitamin C, estrogen and Echinacea, for example. Wetting and lubricating agents such as sodium lauryl sulfate, as well as dyes, flavoring agents, slip agents, fillers, substances for tabletting, stabilizers, antioxidants and preservatives may also be present.

Different carriers and/or excipients suitable for oral administration, which are well known in the art, can be used for the purpose of the invention. The composition, if desired, can also contain various known what s auxiliary substances, such as, for example, preservatives, hardening substances, lubricants, emulsifiers, stabilizers, essence, etc. Such compositions will contain a therapeutically effective amount of the above compounds, preferably in purified form, together with a suitable amount of carrier so as to provide the form for the introduction of the patient. The composition should be suitable to the method of administration.

Usually components are supplied either separately or mixed together in a single dosage form, for example in the form of powder, dried by lyophilization, or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active substance. When the composition should be administered by infusion, it can be dispersed in an infusion bottle containing sterile pharmaceutical grade water or saline.

The pharmaceutical composition of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as anions derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acid, etc. and salts that are formed with cations such as the cations derived from sodium, potassium, ammonium, calcium, iron hydroxides, Isopropylamine, triethylamine, 2-atilim is Netanya, histidine, procaine, etc.

If desired, may be used in vitro to help identify optimal dosage ranges. The exact dose to be used in the composition will also depend on the route of administration and the severity of the disease or of the violation and must be determined in accordance with the decision of the practitioner and circumstances of each patient. Effective doses may be extrapolated from the curves of dependence "dose-effect"obtained on test systems in vitro or in animal models. Preferably the pharmaceutical composition is injected directly or in combination with an auxiliary substance. Excipients can be selected from the group consisting of chloroquine, proton polar compounds such as propylene glycol, polyethylene glycol, glycerin, ethanol, 1-methyl L-2-pyrrolidone or derivatives thereof, or an aprotic polar compounds, such as dimethylsulfoxide (DMSO), diethylsulfoxide, di-n-propylsulfonyl, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylrhodamine, acetonitrile or their derivatives. These compounds are added, given the limitations of the pH level. The composition used in accordance with the present invention, there may be spinal. The term "vertebrate", as used here, implies the same value is, which usually understands by this term an ordinary person skilled in the art. In particular, "vertebral" encompasses mammals, more specifically, people.

The term "entered" means the introduction of a therapeutically effective dose of the above-mentioned composition. The term "therapeutically effective amount" is meant a dose that provides the action for which it is introduced, is preferably anticariogenic effect. The exact dose will depend on the goals of treatment will be determined by the person skilled in the art using known techniques. As is known in the art and described above, may be necessary amendments on the system delivery compared to local delivery, adjustments for age, body weight, General health, sex, diet, time of administration, drug interaction and the many conditions, and they will be determined through routine experimentation by experts in the field of technology.

The methods applied in human therapy and veterinary medicine. Compounds described herein having the desired therapeutic activity, can be entered in a physiologically acceptable carrier to a patient, as described here. Depending on the method of administration, the compounds can be prepared in many forms, as discussed below. The concentration of therapeuti the Eski active compounds in the composition may vary from about 0.1-100 wt.%. Substances may be introduced separately or in combination with other means.

Introduction the pharmaceutical compositions may be implemented in various ways, as described above, including as non-limiting examples of oral, subcutaneous, intravenous, intra-arterial, intra-site, intramedullary, intrathecal, intraventricular, intranasal, intrabronchial, transdermal, vnutriuretralnami, intraperitoneal, intramuscular, intra-lungs, vaginal, rectal or intraocular administration.

Preferably, the introduction is an oral or transbukkalno. Doctor and indicators of clinical status will determine the dosing regimen. As is well known in the field of medicine, dosages for any one patient depends on many factors, including the size of the patient, the surface area, age, the particular input connection, sex, time and route of administration, General health and other drugs administered simultaneously. The usual dose may be, for example, in the range from 0.001 to 1000 μg; however, provided the doses below or above this exemplary range, especially considering the aforementioned factors.

The drug is preferably given once a week, but during treatment, the drug may be given through much what about longer time intervals and, if necessary, may be given through a much shorter time intervals, for example on a daily basis. In the preferred case, the immune response is controlled using the described methods and other methods known to experts in the art, and dosages are optimized, for example, by time, quantity and/or composition. Progress can be monitored through periodic surveys. The pharmaceutical composition of the invention can be injected locally or systemically. Also envisaged that the pharmaceutical compositions are used in combination therapy, i.e. in joint administration with other medicaments or drugs, for example other drugs to prevent, treat or improve caries described here.

In another preferred embodiment, the present invention concerns the use of a microorganism according to the present invention for the preparation of compositions for the treatment and/or prevention of bad breath and/or halitosis, where the composition is a food or feed product and to food or feed products containing the microorganism according to the present invention, its inactivated form, mutant, derivative, analog or fragment. Preferably the composition is in the form of a food or feed product is pexeva is or feed composition, containing the microorganism, its inactivated form, mutant, derivative, analog or fragment described herein above, also containing the orally acceptable carrier or excipient.

"The food or feed product contain any edible with a pleasant taste and/or potable material for mammals, for example humans or animals, such as Pets, as described here. Food and feed product described herein elsewhere. "Orally acceptable carrier" as described here above, and preferably it is non-toxic and food and/or fodder. In addition, the term also covers the media, mentioned in connection with the pharmaceutical composition used in accordance with the present invention.

In accordance with the present invention, the term "food product" includes all edible and potable food and drinks.

Accordingly, the microorganism, its inactivated form, derivative, analog or fragment can be incorporated into food or drink. For example, they are chewing gum, spray, beverage, candy, milk powder, ice cream, frozen dessert, sweet dressing for salad, milk procurement, cheese, quark, lactose-free yogurt, acidified milk, cream for coffee or whipped cream, etc.

OS is consistent milk products included in the scope of the invention. Under the milk, however, it is understood that it is of animal origin, such as cow, goat, sheep, Buffalo, Zebra, horse, donkey or camel, etc. Milk can be in a natural state, resuspending, fat-free or enriched compounds necessary for the growth of bacteria or for further processing fermented milk such as fat, proteins, yeast extract, peptone and/or surfactant, for example. The term "milk" also refers to what is usually called plant milk, and extracts of plant material, which was somehow processed, such as leguminous plants (soybean, chickpea cultural, lentils etc) or oilseed (rapeseed, soybean, sesame, cotton and so on), whose extract contains proteins in solution or in colloidal suspension, which is able to coagulate during the chemical exposure, the acid fermentation and/or heating. Finally, the word "milk" also refers to a mixture of animal and vegetable milk.

When the microorganism of this invention, its inactivated form, or a derivative or analogue or fragment is added to the yogurt, etc. having similar content, is usually sufficient to add the microorganism of this invention at a concentration of about 104-107cells/ml

Such food, drink, Il what food can be produced in the usual way of food and drink or feed, including adding the active ingredient to raw or cooked material food, beverage or feed. Food, beverage or feed according to this invention can be molded and granulomatosa the same way, which is typically used for food, beverage or feed. Methods of molding and granulation methods include granulation, such as granulation in the fluidized bed, whirlpool granulation, extrusion granulation, roller granulation, granulation in the gas flow, granulation by extrusion and molding, crushing granulation, spray granulation, and injection molding granulation, methods of coating, such as lubrication forms a coating in the fluidized bed, and dry coating, drying with blasting method with excess steam, the method using perforated trays, extension methods, such as microwave incubation method, and methods of extrusion extrusion granulation machines and extruders.

Food, beverage or feed of the present invention include any food, drink or food that contain microorganism of the invention, its inactivated form, derivative, or analog, or fragment as an active ingredient. The active ingredient in the food, beverage or feed Conques is to maintain not limited to any concentration, while the finished food product, beverage or food can exercise their activity, expressed in the reduction of unpleasant mouth odor. The concentration of the active component is preferably from 0.001 to 100%, more preferably from 0.01 to 100% and most preferably from 0.1 to 100% by weight of the food, beverage or feed containing such active ingredient, or in relation to the number of cells described here.

Specific foods or drinks, to which is added the active ingredient include, for example, juices, refreshing drinks, soups, teas, fermented milk drinks, dairy products such as fermented milk, ice, butter, cheese, processed milk and skim milk, meat products such as ham, sausage and hamburger, pastries with meat, fish, egg products, such as flavored egg rolls and egg Kurd, confectionery products such as biscuits, jelly, snacks, chewing gum, bread, noodles, pickles, smoked products, dried fish and refills. The shape of the food product or beverage includes, for example, powder products, sheet-like products, bottled products, canned foods, processed in the autoclave, products, capsules, tablets and liquid products.

Food or drink in accordance with the invention, which is accepted by m is adensam, preferably is a food compositions for children. Such food compositions for children include modified milk, prepared for infants milk split protein, special modified nutrient milk, or baby food, or food cooked for the kids. The form of nutritional compositions for infants includes, but is not specifically limited to, powder, dried milk and dried fine and baby food, and also includes conventional products, such as ice cream, fermented milk and jelly for baby food.

Nutritional composition for infants in accordance with this invention mainly consists of protein, lipid, saccharide, vitamins and/or minerals. In the food composition the active ingredient is mixed with these components.

Protein includes proteins of milk, such as skim milk, casein, cheese, whey, whey protein concentrate, whey protein isolate and its fractions, such as alpha s-casein, beta-casein, alpha-lacto albumin and beta-lactoglobulin. Then you can use egg protein, such as protein egg yolk, egg-white protein and egg albumin, or soy protein, such as defatted soy protein, isolated soy protein and concentrated soy protein. Besides, proteins such as wheat gluten, b the Lok meat, fish, protein meat cattle and collagen also acceptable can be used. Then the fraction of these proteins, peptides from their acid or enzyme treatment, or free amino acids also acceptable can be used. Free amino acids can serve as sources of nitrogen and may optionally be used to provide a specific physiological effect. Such free amino acids include, for example, taurine, arginine, cysteine, cystine and glutamine. The lipid includes animal fats and oils, such as milk fat, lard, fat, beef, and fish oil, vegetable oils such as soybean oil, rapeseed oil, corn oil, coconut oil, palm oil, palm kernel oil, safflower oil, perillae oil, Flaxseed oil, primrose oil, medium chain triglyceride fatty acids and cottonseed oil, created by bacteria fats and oils and their fractionated oils, hydrogenated oils and oils obtained by interesterification. The number of lipid, which will be mixed varies depending on the application.

The saccharide includes, for example, one or more of starch, soluble polysaccharides, dextrin, sugars such as sucrose, lactose, as described here, maltose, glucose and fructose, and other oligosaccharides. The total number of such sharedapplication is from 40 to 80 wt.% of the total dry matter content in the food composition. Next, artificial sweeteners such as aspartame, acceptable can be used. The amount of artificial sweetener, respectively, is from 0.05 to 1.0 wt.% of the total dry matter content in the food composition.

Vitamins include, but are not limited to, lycopene as an essential component and optionally include, for example, vitamins such as vitamin a, group b vitamins, vitamins C, D, and E and the group of vitamin K, folic acid, Pantothenic acid, nicotinamide, carnitine, choline, Inositol, and Biotin, if only these vitamins could be administered to infants. These vitamins are preferably ranges from 10 mg to 5 g per the total dry matter content of the nutritional composition for infants.

Further, the minerals include calcium, magnesium, potassium, sodium, iron, copper, zinc, phosphorus, chlorine, manganese, selenium and iodine. Such minerals are preferably from 1 mg to 5 g per the total dry matter content of the nutritional composition for infants.

In addition to those components described above, the nutritional composition for infants in accordance with this invention can be mixed with any component, optionally mixed in food compositions, for example, dietary fiber, nucleotides, nucleic acids, flavorings and colorings.

The food or drink according to izaberete is receiving can be used as a healthy food, or drinks, or functional food or beverage for prevention and/or treatment of unpleasant mouth odor.

When the food or drink according to the present invention taken inside, take the amount is not specifically limited. Accept the amount is usually from 0.1 to 50 g, preferably from 0.5 g to 20 g daily, based on the total amount of the active ingredient.

Food or drink continuously taken by mouth in amounts during the period of time from one day to 5 years, preferably from 2 weeks to one year. Here the received inside the quantity can be adjusted to a suitable range depending on the severity of the symptoms of a person taking inside food or drink, his age and body weight, etc.

Feed according to the present invention may be any food containing the active ingredient. Food includes, for example, feed for domestic animals, particularly dogs, cats and rats, livestock feed, particularly for cows and pigs, feed for chickens and turkeys, and feed for fish farming, in particular for sea bass and yellowtail.

Food can be produced by appropriately mixing the active ingredient described herein above, with the raw feed material, including, for example, the R, cereals, bran, oil cake, raw feed materials of animal origin, other raw feed materials and refined products.

Cereals include, for example, mile, wheat, barley, oats, rye, brown rice, buckwheat, millet, Italian, Chinese millet, cereal judgment, corn and soy.

Bran include, for example, rice bran, low-fat rice bran, bran, flour lowest grade, wheat germ, barley bran, feed waste, corn bran and germ of the corn.

Oilseed meal includes, for example, soybean meal, soy flour, Flaxseed meal, oil cake cotton seed, meal peanut meal safflower, coconut cake, palm oil cake, oil cake sesame, sunflower meal, rapeseed meal, meal Kapok seed and mustard oil cake.

Raw feed materials of animal origin include, for example, fish meal, import, meal, flour of whole grain and flour, concentrated fish broth, meat flour, flour, meat and bone, blood meal, split hair, flour from the bones, by-products from slaughterhouses, flour, bird feathers, pupae of the silkworm, skim milk, casein, whey and krill.

Other raw feed materials include, for example, stems and leaves of plants, such as briquettes Luce the new hay, flour from the leaves of alfalfa and flour from the leaves of lucali, by-products kumarasambhava industry, such as corn gluten meal, corn gluten feed and liquid corn extract, starch, sugar, yeast by-products of industrial fermentation such as beer residue, malt sprouts, sludge liquors and sediment soy sauce, and agricultural by-products such as citrus recycled waste, soybeans, coffee waste and waste cocoa, cassava, horse bean, guar flour, seaweed, spirulina and Chlorella.

Refined products include, for example, proteins such as casein and albumin, amino acids, starch, cellulose, sugars such as sucrose and glucose, minerals and vitamins.

In the case of providing animal feed according to the present invention the quantity of feed is not specifically limited, but is preferably, for example, from 0.1 mg to 50 g per 1 kg of body weight per day, preferably from 0.5 mg to 20 g per 1 kg of body weight per day, based on the amount of the active ingredient. Feed continuously taken during the period of time from one day to 5 years, preferably from 2 weeks to one year. Again, taken orally, the number can be adjusted to a suitable range depending on the species, age and vesatile animal, host inside food, etc.

In another embodiment, the present invention relates to additives to food, drink or feed, comprising a microorganism according to the present invention, its inactivated form, mutant, derivative, analog or fragment, as well as to the use of a microorganism according to the present invention, its inactivated form, mutant, derivative, analog or fragment for the preparation of compositions for the treatment and/or prevention of bad breath and/or halitosis, where the composition is an additive for foods, drinks or feeds. Preferably, the additive to food or drinks includes Supplement nutritional compositions for infants.

Additive to food products can be produced by the usual method of manufacture of additives to food, drink or feed. If necessary, can be added acceptable additives for General use in foods, beverages or feed, for example, additives described in the reference Food Additive Handbook (The Japan Food Additives Association; issued on January 6, 1997), including sweeteners, colorants, preservatives, thickeners and stabilizers, antioxidants, clamps coloring, bleach, antiseptics, gamesnovel, bitters, enzymes, bleaching substances, cyclotourism the tel, refills, emulsifiers, flavor enhancers, tools for production, flavorings and extracts of spices. Next may be acceptable added common sugars, starch, inorganic materials, vegetable powders, fillers, disintegrators, lubricants, binders, surfactants and plasticizers mentioned earlier for pharmaceutical tablets.

Additives include additives.

Sweeteners include aspartame, licorice, stevia, xylose and the fruit of the pumpkin MOMORDICA grosvenori (Momordica grosvenori, rakanka). Colorants include carotenoid and turmeric extract, a flavonoid, caramel coloring, serwisowy dye, chlorophyll, Magenta dye-based sweet potatoes, Magenta dye-based yams, pillowy dye and blueberry dye.

The preservatives include, for example, sodium sulfite, benzoate, benzoin extract, solutes and propionate. Thickeners and stabilizers include, for example, gums, such as Arabian gum and xanthan gum, alginates, chitin, chitosan, aloe extract, guar gum, hydroxypropylcellulose, casein sodium, corn starch, carboxylmethylcellulose, gelatin, agar, dextrin, methylcellulose, polyvinyl alcohol, microfiber cellulose, microcrystalline cellulose, cellulose algae, poliakrilat sodium, the sodium polyphosphate, carrageenan, or the cell wall of yeast.

The antioxidants include, for example, a group of vitamin C, sodium ethylenediaminetetraacetate, calcium ethylenediaminetetraacetate, erythorbate acid, oryzanol, catechin, quercetin, extract of clove, processed by the enzyme rutin, Apple extract, sesame seed extract, dibutylaminoethanol, fennel extract, horseradish extract, aqueous extract of celery, tea extract, Tocopherols, soybean extract, coffee bean extract, sunflower seed extract, Frolovo acid, butylhydroxyanisole, blueberry leaf extract, propolis extract, pepper extract, garden balsam, gallium acid, eucalyptus extract and rosemary extract.

The clips include painting, for example, sodium nitrite. Brighteners include, for example, sodium sulfite.

Antiseptics include, for example, o-phenylphenol. Gamesnow includes, for example, acetylricinoleate methyl, wax lacquer tree (urushi), ester of rosin, elemi resin, wax urumuri, Kauri resin, Carnauba wax, ester of fatty acid and glycerine, wax spermaceti, kopicki balm, copilului resin, rubber, wax rice bran, cane wax, shellac, jelutong, ester of fatty acid and of sucrose, depolimerizovannogo natural rubber, paraffin, fir balsam, ester W the nuclear biological chemical (NBC acid and propylene glycol, powdered cellulose, ground rice hulls, jojoba oil, polyisobutylene, polybutene, microcrystalline wax, mastic resin, beeswax and calcium phosphate.

Bitters include, for example, ISO-alpha-bitter acid, caffeine, extract of kavaratti (Coriolus versieolor)extract, red Peruvian Hinn bark, the bark extract of velvet extract, gentian root, extracts of spices, enzyme modified naringin extract, Cassia Jamaican, theobromine, naringin extract, Cassia extract, absinthe extract of Estonia, olive tea extract, bitter orange (Citrus aurantium)extract of hops, extract of wormwood.

The enzymes include amylase, trypsin or rennin.

Brightening agents include, for example, wax lacquer tree, Japanese wax. Kislotoobrazoutei includes, for example, adipic acid, taconova acid, citric acid, succinic acid, sodium acetate, tartaric acid, carbon dioxide, lactic acid, phytic acid, fumaric acid, malic acid and phosphoric acid. Refills include, for example, amino acids such as asparagine, aspartic acid, glutamic acid, glutamine, alanine, isoleucine, glycine, serine, cysteine, tyrosine, leucine and Proline, a nucleic acid, such as sodium inosinate, uridylate sodium, guanylate sodium, Citadela the sodium, ribonucleotide calcium and ribonucleotide sodium, organic acids such as citric acid and succinic acid, potassium chloride, salt solution with a reduced amount of sodium chloride, raw potassium chloride, salt, whey, tripotassium phosphate, hydrogen phosphate of dicale, potassium dihydrophosphate, disodium hydrogen phosphate, sodium dihydrophosphate, trisodium phosphate and Chlorella.

Flavor enhancers include, for example, a zinc salt, a group of vitamin C, various amino acids, 5-Danilova acid, ferric chloride, hesperidin, various calcined calcium, various ' green ' calcium, dibenzoylethylene, calcium hydroxide, calcium carbonate, thiamin hydrochloride salt, carotene algae, Dunaliella (Dunaliella Carotene, carotene, tocopherol, nicotinic acid, carotene carrots, palm oil carotene, calcium Pantothenate, vitamin a, hydroxyproline, dihidrogenofosfat calcium, ferrous pyrophosphate, pyrophosphate of iron, ferritin, heme iron, menechino, folic acid and Riboflavin.

Tools for production include, for example, an auxiliary means for processing, such as acetone and ion exchange resin. Flavorings include, for example, vanilla essence and extracts of spices include, for example, paprika extract.

These various additives may be added to the active ingredient with which this method of introduction in accordance with this invention.

The composition according to the present invention comprises a microorganism according to the invention. It is envisaged that the compositions contain microorganism in the form of a probiotic microorganism. Namely, in addition to the probiotic effect, the above probiotic microorganism belonging to the group of lactic acid bacteria, are useful for the treatment and/or prevention of unpleasant mouth odor and/or halitosis. The number specified probiotic microorganism is large enough to significantly alleviate the disease that is treated, preferably with bad breath, but low enough to avoid serious side effects (justified the ratio benefit/risk) in the framework of a thorough medical examination. An effective amount specified probiotic microorganism will vary depending on the specific goals you want to achieve, depending on the age and physical condition of the treated patient, the severity of the underlying disease, duration of treatment, nature simultaneously therapy and specific microorganism. An effective amount specified probiotic microorganism, thus, will be the minimum amount that will provide the desired effect. The presence of, for example, 1·109bacteria in the IDA viable or non-viable whole cells in 0.05 ml of a solution of phosphate-saline buffer or 0.05 ml of a suspension of agar, or equivalent dry weight of the fragments of the cell wall, is effective when introduced in quantities of from about 0.05 ml to about 20 ml

The most important practical advantage is that the probiotic organism can be used conveniently, such as oral administration. Depending on the route of administration may require that the active ingredients, which contain the probiotic organisms were covered with material to protect these organisms from the action of enzymes, acids and other natural factors that can inactivate these organisms. To apply probiotic organisms in any way other than parenteral administration, they should be covered or introduced with a material to prevent inactivation. For example, the probiotic organisms can be entered together with enzyme inhibitors or in liposomes. Enzyme inhibitors include pancreatic tripeny inhibitor, diisopropylfluorophosphate (CSP) and trasilol. Liposomes include emulsions, water-in-oil-in-water R40, as well as conventional and special purpose liposomes, which transport the lactic acid bacteria or their by-products to the urogenital surface. Dispersions can also be prepared, for example, in glycerol, liquid polyethylene glycols, and their smese is, and in oils. Typically, dispersions are prepared by incorporating the various sterilized probiotic organisms in sterile carrier medium, which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and drying by sublimation, which give a powder of the active ingredient plus any additional desired ingredient from a previously filtered under sterile conditions a solution of these ingredients. Additional preferred methods of preparation include as non-limiting examples of the lyophilization and heat drying.

The composition also includes products designed for oral or transbukkalno introduction, which contain a pharmaceutically acceptable carrier, as described here, to which or in which the cells of the above mentioned microorganism belonging to the group of lactic acid bacteria are added in a new form, such as concentrated or dried. Of course, can also be added inactivated form, derivative, or analog, or fragment of a specified microorganism, or any combination of the specified microorganism, its derivative, and/or the Academy of Sciences of the log, and/or fragment disclosed here. These products can be provided in the form of the received inside suspension, gel, spray, capsule, hard gelatin capsule, syrup or any other galenical form, well-known specialists in this field of technology.

When probiotic organisms suitably protected, as described above, the active compound may be administered orally, for example, with an inert diluent or with an assimilable edible carrier, or it may be included in hard or soft gelatin capsule, or may be compressed into tablets, intended for passage through the stomach (i.e. enteric-coated shell), or may be incorporated directly with the food. For therapeutic oral administration of probiotic organisms can be incorporated with excipients and used in the form taken orally tablets, tablets for slow dissolution in the buccal pocket, tablets, capsules, elixirs, suspensions, syrups, plates, etc. of the Composition or compositions according to the present invention is prepared so that an oral unit dosage form contains, for example, about 1×109viable or non-viable, for example, lactic acid bacteria per milliliter. Probiotic organism make up for udobnoe effective introduction in effective amounts with a suitable pharmaceutically acceptable or acceptable for consumption by the carrier in unit dosage form, as disclosed above. The unit dosage form can, for example, contain the principal active compound in amounts of approximately 109viable or non-viable, for example, lactic acid bacteria per milliliter. In the case of compositions containing additional ingredients such as prebiotics, dosage determine, based on the usual dose and manner of introduction of these ingredients.

In another embodiment, the present invention relates to a method of prevention and/or treatment of bad breath and/or halitosis. Preferably, the method of prevention and/or treatment includes an introduction to the subject microorganism according to the present invention or inactivated form, or a mutant, derivative, analog or fragment of a specified microorganism described herein above. Preferably, a subject that will be treated, is a beast. More preferably, the animal is a mammal, more preferably a mammal is a mammal pet. In a preferred variant of the invention, the pet is a dog, cat, hamster, monkey, rat or mouse. In another preferred embodiment of the invention the animal is representative of the cattle, horse, pig, donkey, sheep, or to the Oh. In another preferred embodiment the mammal is a human.

The introduction of the microorganism according to the invention in the context of the method of treatment and/or prevention of the present invention may be performed in any suitable form known to the person skilled in the art. Preferably, the introduction covers the use and application of the compositions as described herein above, which can optionally contain, for example, pharmaceutical or cosmetic carriers or excipients, as described here above. Dosage and duration can be set according to any suitable information known to the person skilled in the art. Preferably, the dosage and the period can be installed as described here above.

The invention is illustrated in Figures 1-7, which shows:

FIGURE 1 shows the results of the experiment investigating the effect of reducing the concentration of the peptides of lactic acid bacteria for the production of H2S gram-negative anaerobic bacteria present in the saliva.

FIGURE 2 shows the results of the experiment investigating the effect of supernatants lactic acid bacteria on the growth of S. salivarius, as described in Example 3.

FIGURE 3 shows the results of the experiment investigating the effect Molo is nekislyj bacteria on the growth of R. gingivalis, as described in Example 5.

FIGURE 4 shows the results of the experiment investigating the effect of heat-treated supernatant of lactic acid bacteria on the growth of S. salivarius, as described in Example 6.

FIGURE 5 shows the results of the experiment investigating the reduction of the concentration of the peptides of lactic acid bacteria according to the invention.

FIGURE 6 shows the results of the experiment investigating the reduction of the concentration of peptides liofilizirovannami lactic acid bacteria according to the invention.

FIGURE 7 shows typical amino acid profile of the extract PTU.

A better understanding of this invention and its many advantages will come from the following examples, presented for illustrative purposes only and are not intended for any limitation of the scope of the present invention.

Example 1

'TSY-environment:

'TSY-mix (Difco, USA)30 g/l
Yeast extract (Deutsche Hefewerke,3 g/l
Germany)
MRS lightweight environment:
The peptone of Trypticase:1.0 g/l
Yeast extract:0.5 g/l
Diammonium hydrocitric:0.2 g/l
Sodium acetate:0.5 g/l

gSO4-heptahydrate:0,050 g/l
nSO4-monohydrate:0.025 g/l
D-glucose-monohydrate:1 g/l
To2NRA4:0.2 g/l
Oleic acid:0,1% (wt./about.)
Artificial environment:
Guanine:0.1 g/l
Cytosine:0.1 g/l
Thymidine:0.1 g/l
2'-deoxyadenosine:0.1 g/l
2'-deoxyuridine:0.1 g/l
To2NRA4:2 g/l
Sodium acetate:5 CH
gSO4-heptahydrate:0.1 g/l
Di-ammonium hydrocitric:2 g/l
l2-dihydrate:0.5 g/l
Oleic acid:0,1% (wt./about.)
Cyanocobalamin:0.02 mg/l
Riboflavin:10 mg/l
Folic acid:0.2 mg/l
Pyridoxal-5-phosphate monohydrate:10 mg/l
4-aminobenzoic acid:0.2 mg/l
D(+)-Biotin:1 mg/l
Ascorbic acid:500 mg/l
Nicotinic acid:10 mg/l
CA-Pantothenate:10 mg/l
Thiamine:1 mg/l
Cobalt(II)-nitrate-uranyl:500 mg/l
nSO4-monohydrate: 20 mg/l
gSO4-heptahydrate:500 mg/l
Na2MoO4:0.04 mg/l
PTU-extract (Ohiy, Deutsche Hefewerke, Germany): D-glucose-monohydrate:15 g/l (or as installed in a different location) 10 g/l
FAB environment:
Peptone mixture:15.0 g/l
Yeast extract:10.0 g/l
Sodium thioglycolate:0.5 g/l
Sodium chloride:2.5 g/l
Agar N.1:0.75 g/l
L-cysteine HCl:0.5 g/l
Resazurin:0.001 g/l
Sodium bicarbonate:0.4 g/l
Geminus:0.005 g/l
Vitamin K:0.0005 g/l

Storage and growth

Storage and growth of strains can occur with the use of normal operations. For example, the strains can Ralitsa as frozen raw materials at -80°C. 1 ml of the culture may be grown to stationary phase (OD600/ml 4-8) in the MRS and mixed with 500 µl of sterile 50% solution of glycerol and frozen.

Culture of S. salivarius were grown in medium'TSY to stationary phase (OD600/1-2 ml) and processed as mentioned above. The strain S. salivarius, preferably used in the experiments was a strain of S. salivarius DSM 20560 (Andrews and Horder, 1906).

Cultivation of S. salivarius (DSM 20560), as well as isolates was carried out under anaerobic conditions in a 6-hole tablets with 8 ml of medium'TSY over night at 37°C.

Lactic acid bacteria (DSM 19825, 19826, 19827) were cultivated under anaerobic conditions in 150 μl of the artificial medium in 96-well tablets in 24 hours at 37°C.

Mixing lactic acid bacteria and S. salivarius were carried out in the ratio of cells 1:100 (lactic acid bacteria: S. salivarius)1/2environment'TSY. Mixing was carried out in 96-well plates.

The culture suspension were incubated for 12 hours in a microplate spectrophotometer for BioTek PowerWave at 37°C.

As a control instead of a lactic acid culture used unspent1/2environment'TSY or light environment MRS.

Stimulation of growth of S. salivarius was visible when comparing the maximum optical density (OD600, max) or the maximum growth rate (Vmaxwith or without the lactic acid bacter the th after 10 hours of incubation (OD 600, maxor during exponential growth (Vmax).

Stimulation is increased at least 10% of the maximum optical density (OD600, max) or the maximum growth rate (Vmax).

Example 2

Taxonomic classification of strains

Taxonomic classification of the strains was performed according to the scheme fermentation of carbohydrates. The classification was carried out using the system API 50 CH (bioMarieux, France) and analyzed using the software APILAB PLUS version 3.3.3 (bioMarieux, France).

Example 3

Research to stimulate the growth of Streptococcus salivarius the supernatant of lactic acid bacteria

Bacteria were cultured according to Example 1. The supernatant of the lactic acid bacteria (in particular, DSM 19826) was obtained by centrifugation at 4000·g for 15 min Mixing supernatant of lactic acid bacteria with S. salivarius was performed in a volume ratio of 2:1 to 4:1 (S. salivarius: supernatant of lactic acid bacteria) in1/2environment'TSY. Mixing was carried out in 96-well plates. The culture suspension were incubated for 12 hours in a microplate spectrophotometer for BioTek PowerWave at 37°C. as a control instead of the supernatant of the lactic acid bacteria used unspent1/2environment'TSY or light environment MRS.

Stimulation of growth was evaluated according to Example 1. Can b the lo to see good growth stimulation S. salivarius supernatants lactic acid bacteria. The results are shown in Figure 2.

Example 4

The absence of stimulation of pathogenic representative of the oral flora S. mutans

Culture of S. salivarius were grown according to Example 1. Streptococcus mutans (DSM 20253) were grown in 5 ml of medium'TSY in closed 15-ml Falcon tubes during the night. Bacteria of the oral cavity were mixed in a volume ratio of 2:1 with the supernatant of lactic acid bacteria and estimated growth according to Example 1. As a control instead of the supernatant of the lactic acid bacteria used unspent1/2environment'TSY.

It was possible to observe the absence of any stimulation of the growth of oral pathogenic bacteria S. mutans lactic acid bacteria.

Moreover, the absence of stimulation of the growth of S. mutans can be estimated through the following analysis.

Lactic acid bacteria (for example, DSM 19825, 19826, 19827) were cultivated under anaerobic conditions in 150 μl of the artificial medium in 96-well tablets in 24 hours at 37°C. Streptococcus mutans (DSM 20253) were grown in anaerobic conditions in 5 ml of medium'TSY in closed 15-ml Falcon tubes overnight at 37°C. the Mixing of lactic acid bacteria and S. mutans were carried out in the ratio of cells 1:100 (lactic acid bacteria:S. mutans) in1/2environment'TSY. Mixing was carried out in 96-well plates. The culture suspension Inka is Aravali in aerobic conditions for 12 hours in a microplate spectrophotometer for BioTek PowerWave at 37°C. As a control instead of a culture of lactic acid bacteria used unspent1/2environment'TSY.

Example 5

The absence of stimulation of pathogenic representative of the oral flora P. gingivalis

Culture S. salivanus were grown according to Example 1. Porphyromonas gingivalis (DSM 20109) were grown in anaerobic conditions in 5 ml of medium FAB in closed 15-ml Falcon tubes at 37°C over night. P. gingivalis was mixed in a volume ratio of 2:1 with the supernatant of lactic acid bacteria (from DSM 19826) and cultivated under anaerobic conditions in 96-well plates. As a control instead of the supernatant of the lactic acid bacteria were cultivated P. gingivalis still FAB environment.

No stimulation of growth of oral pathogenic bacteria P. gingivalis lactic acid bacteria was not observed. The results are shown in Figure 3.

Moreover, the absence of stimulation of the growth of P. gingivalis can be estimated through the following analysis.

Lactic acid bacteria (for example, DSM 19825, 19826, 19827) were cultivated under anaerobic conditions in 150 μl of the artificial medium in 96-well tablets in 24 hours at 37°C. Porphyromonas gingivalis (DSM 20709) were grown in anaerobic conditions in 5 ml of medium FAB in closed 15-ml Falcon tubes overnight at 37°C. the Mixing of lactic acid bacteria and P. gingivalis were carried out in the ratio of cells 1:100 (the breast is okolie bacteria:P. gingivalis) in the FAB environment. Mixing was carried out in 96-well plates. The culture suspension were incubated aerobically for 45 hours in an anaerobic workstation Whitley DG250 (Meintrup-DWS, Germany) at 37°C. as a control instead of a culture of lactic acid bacteria used unspent Wednesday FAB.

Example 6

Temperature stability stimulating ability of lactic acid bacteria

Bacteria were grown according to Example 1. Supernatant lactic acid bacteria (DSM 19827) were incubated at 80°C for 10 min in the incubator. After cooling, the supernatant to room temperature, the supernatant of the lactic acid bacteria were mixed in a volume ratio of 1:2 with grown cultures of S. salivarius and evaluated the stimulation according to Example 1, including control experiments.

(Stimulation was assessed using oral pathogenic bacteria, as broadly described in Examples 4 and 5. It was shown that nelinumeroisia behavior of lactic acid bacteria against oral pathogenic bacteria not affected by heat treatment.)

It was possible to observe the absence of the influence of thermal processing on the stimulatory activity against S. salivarius. The results are shown in Figure 4.

Example 7

Sensitivity to stimulation by lyophilization

S. salivarius were grown according to Example 1. Moloch is acid bacteria were cultivated under anaerobic conditions in 50 ml of artificial medium in 100-ml flasks (Schott, Germany) over night at 37°C. the Supernatant of the lactic acid bacteria was obtained by centrifugation at 4000×g for 15 min to 20 ml of the supernatant were frozen to -80°C and liofilizirovanny in vacuum for 16 hours. Freeze-dried supernatant resuspendable in 20 ml of H2O. Resuspending supernatant was mixed with a culture of S. salivarius in the ratio 2:1 (S. salivarius:resuspending supernatant) in1/2environment'TSY in 96-well plates. Growth stimulation was evaluated according to Example 1, including control experiments.

Stimulating activity was not increased during lyophilization.

Example 8

The decrease in the concentration of peptides using lactic acid bacteria

Lactic acid bacteria (DSM 19827) were cultured according to Example 1. The main part of the culture was cultured in an artificial medium containing 15 g/l of peptides (extract PTU). Wednesday was inoculable 10 ál of the culture suspension was cultured under anaerobic conditions at 37°C for 24 hours. Then determined the concentration of peptides and found that it decreased at least 20% after 24 hours. The results demonstrated effective reduction in the concentration of peptides growing lactic acid bacteria. The results are illustrated in Figure 5.

Example 9

Sensitivity decrease in the concentration of peptides to lyophilization

Lactic acid bacteria (DSM 19827) were cultured in 100 ml of artificial medium at 37°C for 24 hours. The entire culture was centrifuged at 4000·g for 15 min and resuspendable in 20 ml of H2O. 20 ml respondieron lactic acid bacteria were frozen to -80°C and liofilizirovanny in vacuum for 16 hours.

For analysis of spending peptides 10 mg of lyophilized lactic acid bacteria resuspendable in H2O and centrifuged at HD within 10 minutes 1 ml of artificial medium containing 7 g/l peptides were added to the precipitate and after 5 min incubation at 37°C. the cells were removed by centrifugation. Determined the concentration of peptides in the supernatant. The concentration of peptides in the medium after removal of the cells was reduced to 2 g/L. This corresponds to the expenditure of 0.5 mg peptide/mg of lyophilized lactic acid bacteria.

These results show effective reduction in the concentration of the peptides of lactic acid bacteria in dried condition, which is illustrated in Figure 6.

Example 10

The effect of lowering the concentration of peptides using lactic acid bacteria for the production of H2S gram-negative anaerobic bacteria

Lactic acid bacteria were cultured and liofilizirovanny according to Example 8. For the experiment, 10 mg of lyophilized lactic acid bacteria resuspending is whether H 2O globalinvacom tablet and centrifuged at 4000·g for 10 min, 1 ml of the artificial medium containing 3 g/l peptides were added to the precipitate and after 5 min incubation at 37°C. the cells were removed by centrifugation. The pH of the medium did not change during incubation.

The environment then was inoculable 50 ál of sterile human saliva and incubated under anaerobic conditions for 6 hours at 37°C. Globaloney tablet covered with sterile filter paper impregnated with lead acetate. The production of hydrogen sulfide by microorganisms from saliva controlled tracking blackening of the filter paper.

Watched reduced production of N2S in the medium treated with lactic acid bacteria, comparing blackening with the control experiment without prior incubation with lactic acid bacteria.

These results show that pre-incubation medium with lactic acid bacteria leads to reduced production of H2S during subsequent incubation medium with microorganisms from human saliva.

Example 11

The composition of lozenges (I)

Composition of tablets for sucking preferably prepared as described in Example 4 on page 8 of the German patent DE-C2 3645147, where in addition to the components mentioned in the Example 4, Viseu amenity microorganism, belonging to the genus of lactic acid bacteria is added in an amount of from 102up to 1012preferably from 103up to 108cells on mg lozenges.

Example 12

The composition of lozenges (II)

Composition of tablets for sucking preferably prepared as described in Example 5 on page 8 of the German patent DE-C2 3645147, where in addition to the components mentioned in the Example 5, the above mentioned microorganism belonging to the genus of lactic acid bacteria is added in an amount of from 102up to 1012preferably from 103up to 108cells on mg lozenges.

Example 13

Composition for cleaning teeth

Composition for cleaning teeth preferably prepared as described in Example 3 on page 8 of the German patent DE-C2 3645147, where in addition to the components mentioned in the Example 3, the above mentioned microorganism belonging to the genus of lactic acid bacteria is added in an amount of from 102up to 1012preferably from 103up to 108cells on the mg tools for cleaning teeth.

Example 14

Composition means for brushing on the basis of a chalk

Composition means for brushing on the basis of a chalk preferably prepared as described in Chapter 7.1.4.4. "Rezepturbeispiel" str guide "Kosmetik", W Umbach (editor), 2nd edition, Thieme Verlag, 1995, where in addition to the components mentioned in this Chapter on str, the above mentioned microorganism belonging to the genus of lactic acid bacteria is added in an amount of from 102up to 1012preferably from 103up to 108cells on the mg tools for brushing on the basis of chalk.

Example 15

Gel for brushing on the basis of silicic acid/sodium fluoride

Composition gley means for brushing on the basis of silicic acid/sodium fluoride is preferably prepared as described in Chapter 7.1.4.4. "Rezepturbeispiel" str guide "Kosmetik", W. Umbach (editor), 2nd edition, Thieme Verlag, 1995, where in addition to the components mentioned in this Chapter on str, the above mentioned microorganism belonging to the genus of lactic acid bacteria is added in an amount of from 102up to 1012preferably from 103up to 108cells per mg of gel means for brushing on the basis of silicic acid/sodium fluoride.

Example 16

Composition for cleaning teeth against plaque

Composition for cleaning teeth against plaque preferably prepared as described in Chapter 7.1.4.4. "Rezepturbeispiel" str guide "Kosmetik", W. Umbach (editor), 2nd edition, Thieme Verlag, 1995, where in addition to the components mentioned in this Chapter on str, videopornocaserogratis, belonging to the genus of lactic acid bacteria is added in an amount of from 102up to 1012preferably from 103up to 108cells on the mg tools for brushing your teeth against plaque.

Example 17

The composition of a chewing gum

The chewing gum composition is preferably prepared as described in Example 6 on page 9 of German patent DE-C2 3645147, where in addition to the components mentioned in the Example 6, the above mentioned microorganism belonging to the genus of lactic acid bacteria is added in an amount of from 102up to 1012preferably from 103up to 108cells per mg of chewing gum.

Example 18

The composition of concentrated mouthrinse

The composition of concentrated mouthrinse preferably prepared as described in Chapter 7.1.4.4. "Rezepturbeispiel" str guide "Kosmetik", W. Umbach (editor), 2nd edition, Thieme Verlag, 1995, where in addition to the components mentioned in this Chapter on str, the above mentioned microorganism belonging to the genus of lactic acid bacteria is added in an amount of from 102up to 1013cells / mg of concentrated mouthrinse.

Example 19

Film preparation

Preparation of films:

1) aqueous phase

- water is heated to 60°C

- aspartame (sweetener) add at premesis the research Institute

- aspartame is completely dissolved.

- polymeric water-soluble film-forming material, such as Kollicoat IR (a polyethylene glycol and polyvinyl alcohol), or PVP (polyvinylpyrrolidone), or natural polymers such as alginates, add stirring until they dissolve

after 10 min the rest of the film is removed

- the above mentioned microorganism belonging to the genus of lactic acid bacteria is added in an amount of from 102up to 1012preferably from 103up to 108cells are ready aromatic film added after cooling the mixture; alternatively, you can add a mutant or derivative of the above mentioned microorganism belonging to the genus of lactic acid bacteria, or similar, or a fragment of the above mentioned microorganism belonging to the genus of lactic acid bacteria

2) the oil phase

- dissolve menthol in peppermint oil

- Polysorbate 80 was added with stirring to a mixture of peppermint oil and menthol

- this mixture is then added with stirring to the propylene glycol

- you can add an optional colorants (such as pigments, varnishes)

- when mixing the oil phase is slowly mixed with the aqueous phase

- thin film mechanically produced using the cutter

Other vari the details of the implementation and enforcement of the invention will be obvious to a person skilled in the art from the description and analysis of the application of the invention, unveiled here. All here for any reason, including all publications, all U.S. patents and other countries and all U.S. patent applications and other countries, and specially in its entirety is included here by reference for all purposes. It should be understood that the description and examples are illustrative only valid in the scope and nature of the invention indicated by the following claims.

1. A means to reduce unpleasant mouth odor, representing strains of microorganisms Lactobacillus acidophilus selected from the group of: Lactobacillus acidophilus DSM 19825, Lactobacillus acidophilus DSM 19826 and Lactobacillus acidophilus DSM 19827.

2. The tool according to claim 1, characterized in that it additionally is able to stimulate the growth of Streptococcus salivarius, but does not stimulate the growth of Streptococcus mutans and/or Porphyromonas gingivalis.

3. The tool according to claims 1 or 2, representing strains of microorganisms Lactobacillus acidophilus in inactivated form.

4. The use according to one of claims 1 to 3 to reduce unpleasant mouth odor.

5. Composition to reduce unpleasant mouth odor, contains a tool according to one of claims 1 to 3 in an amount of from 102up to 1012 cells / mg of composition, or in the range from 102up to 1013cells per ml of composition.

6. The composition according to claim 5, where this composition is a means for brushing your teeth, chewing gum, tablet dispersible, mouthrinse, mouthwash, dental floss or dental tape.

7. A means to reduce unpleasant mouth odor, representing the culture supernatant of strains of microorganisms Lactobacillus acidophilus selected from the group of: Lactobacillus acidophilus DSM 19825, Lactobacillus acidophilus DSM 19826 and Lactobacillus acidophilus DSM 19827.

8. The use according to claim 7 to reduce unpleasant mouth odor.

9. Composition to reduce unpleasant mouth odor, contains a tool according to claim 7 and a carrier.

10. The composition according to claim 9, where this composition is a means for brushing your teeth, chewing gum, tablet dispersible, mouthrinse, mouthwash, dental floss or dental tape.



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of microbiology and deals with method of obtaining RTX-toxin ApxI. Claimed method is realised by cultivation of bacteria Actinobacillus pleuropneumoniae in culture medium, which provides growth of bacteria, and said culture medium contains borogluconate in concentration lower than 60 mmol/l in order to form in medium calcium-bologluconate complex.

EFFECT: invention makes it possible to increase output of RTX-toxin ApxI, which can be applied in production of vaccines.

5 cl, 4 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to produce thermolabile enterotoxin (LT-enterotoxin) and Hafnia alvei anatoxin when producing a vaccine. The strain is deposited in the State Collection of Pathogenic Microorganisms of FBSI Scientific Centre for Evaluation of Medical Products of the Ministry of Public Health and Social Development of Russia under number 294.

EFFECT: strain has high capacity for producing thermolabile LT-enterotoxin.

1 tbl, 2 ex

FIELD: biotechnology.

SUBSTANCE: biological preparation comprises the culture fluid containing the strain of nitrogen-fixing nodule bacteria with a titre of not less than 107 CFU/ml, and an aqueous solution containing (wt %): sodium carboxymethyl cellulose (2.0-5.0), potassium sorbate (0.5-3.0) and glycerol (1.0-5.0), mixed in a ratio of from 1:1 to 1:2.

EFFECT: invention enables to increase the productivity of leguminous crops and to increase the shelf life of the resulting preparation.

3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to microbiology, particularly to the protection of agricultural, medicinal and forest crops. The strain Bacillus thuringiensis var. thuringiensis No.800/15 (BtH1 800/15) possesses the entomocidal activity on depredators - phytophage insects. The strain is deposited in State Scientific All-Russia Research Institute for Agricultural Microbiology (GNU VNIISHM), No. 611.

EFFECT: strain may be used in preparing the high-efficacy entomocidal biopreparation.

3 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: strain Bacillus thuringiensis var. darmstadiensis №25 (BtH10 №25) has insecticidal activity against pests - coleopteran insects and a wide range of antifungal activity against pathogenic fungi. It is deposited in State Scientific Institution All-Russia Institute for Agricultural Microbiology under the registration number RCAM01490. It can be used in the manufacture of polyfunctional means of protection of plants against harmful coleopteran insects and phytopathogenic fungi.

EFFECT: invention also enables to improve the germinating capacity of plants.

7 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: strain of bacteria Bacillus atrophaeus VKPM V-10592 is grown, and suspension is prepared from it, which is introduced into permafrost soil and water environment. Maintained at the specified parameters from 7 to 60 days, and then they determine quantity content of oil and oil products in permafrost soil and water environment.

EFFECT: invention makes it possible to reduce time for oil and oil product denaturation and to reduce their concentration in permafrost soil and water environment.

5 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining androst-4,9(11)-dien-3,17-dione from phytosterol. Microbiological oxidative elimination of side chain at atom C17 with formation of 9α-hydroxyandrost-4-en-3,17-dione is performed. Biomass is separated. 9α-hydroxyandrost-4-en-3,17-dione is extracted from clarified cultural liquid with aprotic organic solvent, selected from aromatic hydrocarbons or organochlorine hydrocarbons. After that, reaction of 9α-hydroxygroup of 9α- hydroxyandrost-4-en-3,17-dione dehydration is carried out in obtained extract. As dehydration agent applied is mineral acid, which contains water and is selected from group, which includes orthophosphoric, pyrophosphoric and chloric acids. Mineral acid is applied in quantity from 1 to 10 mol per 1 mol of 9α- hydroxyandrost-4-en-3,17-dione. In the process of dehydration reaction removal of excessive water is carried out either in presence of effective quantity of pyrophosphoric acid or by azeotropic distillation.

EFFECT: invention makes it possible to intensify dehydration process with application of smaller quantity of mineral acid and exclude side product formation.

11 cl, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, microbiology, and concerns the recovery and identification of pseudotuberculosis and intestinal yersiniosis agents (Y. Pseudotuberculosis and Y. Enterocolytica). A nutrient medium contains microbiological agar (dry), lactose, glucose, urea, calcium chloride, 1% alcoholic phenol red, 1% alcoholic methylene blue and distilled water in specific proportions.

EFFECT: invention enables reducing the length of studies.

3 dwg, 2 tbl

FIELD: biotechnology.

SUBSTANCE: growing of Staphylococcus aureus is carried out in a nutrient medium containing yolk-salt agar. At a stage of preparation for analysis the growth stimulators of Staphylococcus aureus are introduced into the nutrient medium in the form of aqueous solutions at concentrations of 10-4-10-6 wt %. The following compounds are used as growth stimulators: tris(2-hydroxyethyl)ammonium 4-chlorophenyl-sulfanylacetate or tris(2-hydroxyethyl)ammonium 2-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 2-methyl-4-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 1-benzylindol-3-yl-sulfanylacetate.

EFFECT: invention enables to accelerate growing of Staphylococcus aureus for diagnostics of infections, by reducing the time of growing from 48 to 6-9 hours in comparison with the control in a standard nutrient medium.

2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of removing pesticides from Ginkgo biloba extracts. The method involves subjecting an extracted obtained by extracting Ginkgo biloba leaves with hexane, comprising the following steps: a) liquid-liquid extraction to obtain a medium polarity fraction containing ginkgo terpenes and pesticides not removable with hexane, and a high polarity fraction containing ginkgo flavone glycosides; b) crystallisation from the medium polarity fraction obtained at step a) to form an intermediate with ginkgolide content of not less than 50%; c) crystallisation from the residual solution obtained at step b) to obtain an intermediate product with bilobalide content of not less than 50%; d) mixing of the high polarity fraction obtained at step a) with the ginkgo terpene intermediate product and the bilobalide intermediate product obtained at steps b) and c).

EFFECT: invention enables to obtain a product with a given ratio of terpenes, in which residual content of pesticides is considerably low or pesticides are completely removed.

4 cl, 1 tbl, 5 ex

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