Health-promoting composition and method for preparing it

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to health-promoting compositions and methods for preparing them. A method for preparing the composition of non-living lactic acid bacilli, possessing an ability of specific binding to Streptococcus mutans, involves the following stages: heating a cell suspension of lactic acid bacillus or a mixture of lactic acid bacilli possessing an ability of specific binding to Streptococcus mutans from an initial temperature of less than 40°C to a pasteurisation temperature of 75 to 85°C with a temperature variation within the range of 0.5 to 2°C/min, keeping the heated suspension at a pasteurisation temperature of 20 to 40 minutes and cooling the suspension to a final temperature of less than 40°C within the range of 0.5 to 2°C/min. The specific binding the cell suspension of the lactic acid bacillus or the mixture of lactic acid bacilli to Streptococcus mutans is stable to heat treatment and/or resistant to proteases and/or calcium-dependent and/or is observed within the range of pH values falling within the range of 4.5 and 8.5, and/or in the saliva environment.

EFFECT: invention enables producing the agent preventing or delaying the caries lesion formation.

9 cl, 4 ex

 

The invention relates to the field of healthy compositions and method of their preparation.

With the development of caries Central role played by the bacterium Streptococcus mutans. This bacterium transforms are capable of fermenting sugars to organic acids, thus creating an acidic microenvironment. Organic acids can demineralizing tooth enamel and thereby cause the development of carious lesions or promote him. In addition, S. mutans form insoluble glucan matrix. The latter contributes to the formation and adhesion of dental plaque, as well as the adhesion of S. mutans to the tooth surface. It further found that in carious lesions often there are other bacteria, but always together with S. mutans. Therefore, at present, it is assumed that the presence of S. mutans is a mandatory prerequisite for the development of carious lesions.

There is a constant need for means by which it is possible to prevent the formation of carious lesions. In this regard, the object of the invention is to provide means by which you can affect S. mutans to prevent, stop or slow the formation of carious lesions. In addition, the object of the invention is to provide a method of obtaining such funds.

To combat carious damage�mi, in particular, to prevent or at least retard their education, in the past he experienced a variety of means. In particular, the specialist known conventional chemical means for combating dental caries and associated with caries microorganisms used in toothpastes, the compositions of mouthwash and/or other means to care for your teeth. In addition, attempts were made to deal with associated with caries organisms with other organisms or their products, in particular, reducing the number of teeth or in the mouth, or by other impact on their cariogenicity. Such an approach is described for example in application WO 2006/027265 A1. However, its disadvantage is that in many cases the use of living microorganisms in the care mouth consumers will not be accepted or completely prohibited by the legislature. In addition, the products of metabolism of living organisms can affect the taste and/or appearance of the containing product, and this effect may be undesirable in some products to combat carious lesions. In addition, the use of living microorganisms in foods to combat carious lesions limits the shelf life of these products.

Therefore, in General, in different areas of technology instead of trying live microorgan�of smov use a metabolically inactive microorganisms, in particular lyophilized microorganisms. The problem is that such microorganisms in favorable conditions can again become metabolically active, for example, if they get them in a favorable aquatic environment. Therefore, the use of metabolically inactive microorganisms associated with significant restrictions regarding the possible composition containing product.

So in other technology areas, attempts were made to produce products with womersleyi microorganisms instead of products with live or metabolically active microorganisms. For example, in application WO 01/95741 A1 describes a food product to help balance intestinal containing viable lactic acid bacteria. Lactic acid bacteria can be deprived of vitality by heat treatment, for example, pasteurization or sterilization. In the document, however, also pointed to the risk that due to heat treatment can be lost and some otherwise achieved health promoting effects of microorganisms. Although the document States that these contribute to the health effects can be eliminated by "selectively", there is no guidance on how you can avoid losing the desired conducive to the health of the symptom during thermal processing�e of microorganisms. Thus, on the basis of this document specialist cannot sensibly predict or imagine what promotes health effects are lost during heat treatment of microorganisms and the effects are not lost. In particular, it is not predictable or foreseeable, is lost anticaries effect due to thermal processing.

Therefore, the object of the invention is to provide means for the suppression of carious lesions, in particular, the means to prevent or slow the formation of carious lesions. In particular, funds should achieve the desired effect. Their production should be simple and inexpensive, and if possible the above shortcomings should be absent or present only to a small extent.

Thus, the invention relates to a method for producing inanimate composition of lactic acid bacteria with the ability of specific binding to Streptococcus mutans, comprising the following stages:

(i) heating a suspension of cells of lactic acid bacteria or a mixture of lactic acid bacteria, capable of specific binding to Streptococcus mutans and the specific binding is

(a) resistant to heat treatment and/or

b) resistant to treatment with protease and/or

in) dependent on calcium and/or

d) origin�odit in the range of pH values between 4.5 and 8.5, and/or

d) occurs in the presence of saliva,

with the initial temperature below 40°C up to the pasteurization temperature of 75 to 85°C with temperature variation from 0.5 to 2°C/min

ii) holding the heated slurry at pasteurization temperature for 20 to 40 min,

iii) cooling the suspension with stage (ii) to a final temperature below 40°C with temperature variation from 0.5 to 2°C/min.

Such lactic acid bacteria are disclosed in the application WO 2006/027265 A1, the contents of which are fully referenced for the purpose of disclosing the present invention.

Unexpectedly, it was found that by careful pasteurization selected according to the invention the lactic acid bacteria according to the stages of the proposed method it is possible to obtain a composition completely or mostly free living, metabolically active microorganisms, lactic acid bacteria, but capable of specific binding to Streptococcus mutans. Therefore, obtained according to the invention a composition suitable for creating anticariogenic effect and, therefore, as a means to prevent and/or treat caries. The concept of "decay" and "carious damage" within the present application are used interchangeably and denote a chronic infectious disease characterized by the formation of softened places, respectively in the tooth, and pureprogressive leading to death of the tooth. Caries can be diagnosed by known methods, and the specialist may refer, for example, to the publication of Angmar-Mansson and ten Bosch, "Adv. Dent. Res." No. 7 (1993), 70 to 79.

In the framework of the present invention, the term "fight against tooth decay", respectively "wrestling with carious lesions" also includes the prevention of dental caries. That is, the face, the mouth of which should be free from S. mutans, can benefit from the achieved according to the invention compositions in the sense that these songs are linked by accidentally falling S. mutans cells and facilitate their elimination.

In the framework of the present invention, the term "caries treatment" also refers to the country obtained according to the invention compositions are suffering from decay the patient to reduce the number of S. mutans cells and removal of S. mutans from the mouth, in particular throughout the oral cavity, including the teeth and interdental spaces.

Selected according to the invention the microorganisms are very heat-resistant with respect to their ability of binding to S. mutans. This allows you to retain described in the application WO 2006/027265 A1 for living cells of lactic acid bacteria that contribute to the health effect of binding to S. mutans after pasteurization according to the invention, not losing it. This is particularly unusual, because in principle it should be assumed that during pasteurization microorgani�MOU denaturised a significant proportion, for example, proteins, and are decomposed or damaged other components of cells that contribute to the health effects of microogranisms usually lost after pasteurization. And in the aforementioned application WO 01/95741 A1 indicates only that in the case of certain lactic acid bacteria may save health promoting effect on the intestinal flora in spite of pasteurization. However, the mouth has a very different structure, contents and load than the intestine, so that the expert could not bear the technical solution according to the aforementioned document to the present invention.

A further object of the invention is a composition of lactic acid bacteria with the ability of specific binding to Streptococcus mutans, received or obtained by the proposed method is received. The composition has the above advantages of the production method according to the invention, in particular it is suitable for the production of anticariogenic product for use on the human body.

Preferably the cells of lactic acid bacteria selected from cells of strains Lactobacillus paracasei, Lactobacillus rhamnosus, or their mixtures. The method of producing according to the invention can be carried out using a suspension of a pure culture of the strain of lactic acid bacteria, in particular strains of Lactobacillus paracasei or Lactobacillus rhamnosus, or a mixture of two, three, even�Rech, five, six, seven or more strains. Especially preferred for the method according to the invention are mentioned in the application WO 2006/027265 A1 species and strains of lactic acid bacteria, in particular those numbers DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 and DSMZ 16673 deposited in the German collection of microogranisms and cell cultures, address of Macerater Ber 1B, Braunschweig, Germany, 26 August 2004, When in the framework of the present invention it comes to cells of lactic acid bacteria, for example, at least preferably, cells of the mentioned strains and mixtures of the two, three, four, five, six or seven of these strains. Specialist it is obvious that the method according to the invention instead of cells, or in addition to the cells of one of ukazannyh strains can also be used a mutant or derivative cell line, and a mutant or derivative cell lines retained the ability of specific binding to S. mutans.

The cells are preferably lactic acid bacteria have the ability of specific binding to Streptococcus mutans serotype C (DSMZ 20523) and/or serotype e (NCTC 10923) and/or serotype f (NCTC 11060).

Mutant, in particular a mutant of one of the above strains of lactic acid bacteria, has one or more immutable genetic changes, it is usually nucleic acids� (acids). Such changes typically include point mutations, such as transition or transverse, as well as a deletion, insertion and joining one or more of the bases of nucleic acids, whereby nucleic acid is modified, and is caused by aberrant gene expression and/or transcription and/or translation, respectively inactivation of gene products. Mutations can occur spontaneously or due to exposure to agents such as, for example, chemicals or radiation. Ways of selecting and obtaining mutants and derived cell lines are described, for example, Sambrook, "Molecular cloning, a laboratory manual" ["Molecular cloning, a guide for the laboratory"], Cold Spring Harbour Laboratory, new York, 2001, and Ausubel, "Current protocols in molecular biology" ["Current protocols in molecular biology", Green Publishing Associates and Wiley Interscience, new York, 1989 Further information specialists in the application WO 2006/027265 A1.

In the framework of the present invention, the concept of "specific binding" respectively "the ability of specific binding to Streptococcus mutans means used according to the invention the cells of lactic acid bacteria, respectively pasteurized according to the invention, the cells are contacted with S. mutans, but not in contact with the majority, or not in contact with any, other microorganisms, and usually�audica in the oral cavity in a significant number. Preferably selected according to the invention, the microorganisms do not bind to the cells of Streptococcus salivarius, Streptococcus salivarius thermophilus, Streptococcus oralis, Streptococcus mitis and/or Streptococcus sanguinis. Particularly preferably used according to the invention the cells of lactic acid bacteria do not bind to Streptococcus salivarius ssp.thermophilus API 50 CH (Biomerieux, France), Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and/or Streptococcus sanguinis DSMZ 20567. Further, as used according to the invention the cells of lactic acid bacteria is preferably not in contact with bacteria of other genera than streptococci. Especially preferably, they are not associated with Staphylococcus epidermidis DSMZ 1798 and/or Stapyhlococcus epidermidis DSMZ 20044. To test the ability of the associate specialist does as described in the application WO 2006/027265 A1 method, and it mixes named bacteria with selected according to the invention, lactic acid bacteria, respectively their pasteurized residues in a volume ratio of 3:1 and observes possibly caused by lactic acid bacteria aggregation. The appropriate method described in example 3 of the mentioned international application.

In accordance with this preferred method, according to which cells of one or more preferred strains of lactic acid bacteria, in particular, L. paracasei or L. rhamnosus, and preferably od�CSOs or more of strains DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 and DSMZ 16673 and especially preferably at least DSMZ 16671 and/or the above-described mutant or derivative cell line, in the first stage is heated in suspension from the source temperature below 40°C up to the pasteurization temperature of 75 to 85°C with temperature variation from 0.5 to 2°C, in the second stage within 20 to 40 min, hold at temperature pasteurization (time pasteurization) and in the third stage, the suspension is cooled to a final temperature below 40°C with temperature variation from 0.5 to 2°C.

The pasteurization temperature in stage (i) is preferably 78 to 80°C., particularly preferably 80°C. At this temperature pasteurization provided fast and energy-saving, and reliable killing selected according to the invention the cells of lactic acid bacteria while a slight decrease in the binding ability compared to unpasteurized culture of living cells of lactic acid bacteria.

The pasteurization time is preferably 25 to 35 min, particularly preferably 30 minutes. It was found that in particular at pasteurization temperature from 78 to 80°C. (preferably 80°C) for quicker killing selected according to the invention the cells of lactic acid bacteria in a small loss of ability the binding of S. mutans compared with the culture alive�x cells of lactic acid bacteria.

Further preferably, the temperature change at stages (i) or (iii) is 0.8 to 1.2°C/min, preferably 1°C/min, which provides a short heating to the temperature of pasteurization and therefore saves energy without compromising the reliability of killing of cells of lactic acid bacteria and without severe loss of the ability of binding to S. mutans.

Particularly preferred is a method in which the cells of one or more preferred strains of lactic acid bacteria, L paracasei or L. rhamnosus, preferably one or more of strains DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 and DSMZ 16673, particularly preferably at least DSMZ 16671, and/or the above-described mutant or derivative cell line, in the first stage is heated in suspension from the source temperature below 40°C up to the pasteurization temperature from 78 to 80°C, preferably 80°C, when the temperature changes from 0.8 to 1.2°, preferably 1°C, in the second stage for 25 to 35 minutes, preferably 30 minutes, hold at temperature pasteurization (pasteurization), and the suspension in the third stage is cooled to a final temperature below 40°C with temperature variation from 0.8 to 1.2°, preferably 1°C.

Pasteurized according to the invention, the cells are preferably in postexperimental the growth phase. Especially preferred�Stateline on stage (i) the proposed method of obtaining the use of cells from culture media, which ensured the exponential growth, in which, however, the glucose concentration fell to not more than 1 mmol / l glucose. It is preferable to use cells that were at the level of glucose concentration to a maximum of 1 mmol for not more than one hour, otherwise it begins to lose the capacity obtained by the proposed method product to specific binding to Streptococcus mutans, i.e. the product obtained in stage (iii), and, if required, at the stage of washing or spray drying, as described below.

Further, the method according to the invention preferably includes a step of washing, in which step (iii) the suspension was centrifuged to increase the concentration of pasteurized cells thus obtained concentrated cells are mixed with water and again centrifuged to increase the cell concentration. The obtained concentrated cells are available as mass, in which the dry matter content is from 10 to 30%, preferably from 18 to 22% by weight of the mass.

Especially preferred is a method, along with the stages (i) to (iii) (in particular, using one strain of lactic acid bacteria or a mixture of two, three, four, five, six or seven strains of lactic acid bacteria selected from DSM 16667, DSM 16668, DSM 16669, DSM 16670, DSM 6671, DSM 16672 and DSM 16673, or using the above-described mutant or derivative cell line or strain of L. paracasei or L. rhamnosus) and, if necessary, rinsing, comprising the following stages:

iv) mixing of step (iii) and preferably washed suspensions with medium to obtain a mixture for spray drying, wherein the carrier is selected from sulfates of alkali and alkaline earth metals, preferably of sodium sulfate, potassium or calcium, and mixtures of two or more of these sulfates, and wherein the amount of the carrier selected with providing the content of dry matter in the mixture for spray drying from 10 to 30 wt.%, and

v) spray drying of step (iv) mixture for spray drying.

In spray drying to be dried material is subjected to very harsh conditions, particularly the temperature from 70°C to 200°C., Until now it was not known that is selected according to the invention the cells of lactic acid bacteria can withstand the harsh conditions of the spray drying without significant loss of the ability of specific binding to S. mutans. In addition, due to the large proportion of media according to the invention at the stage iv) would be expected that responsible for the binding of S. mutans to the surface structure of lactic acid bacteria denature or otherwise damaged. Unexpectedly it was found, �when the selected conditions are possible spray drying of step (iii) suspension without significant loss of the ability of binding to S. mutans.

Some of the advantages of spray drying according to the invention and obtained as a result of the present composition deserve special attention. Unlike simple pasteurization, spray drying according to the invention provides getting sticky or impervious composition. The term "hygroscopic" in this context means that with open storage at 20°C for 12 months and at a relative humidity of the air component of 50%, the weight of the composition increases by no more than 2%. The thus obtained composition according to the invention are particularly suitable for further processing into dry products, it does not adversely affect the properties of these products due to the attraction of water or special about this item.

Further, the proposed method of producing provides a composition according to the invention, which is freely flowing. Storage and processing of free flowing compositions in the form of particles are particularly simple, and such compositions can be administered in different products, and with a precise dosage. Thanks to that obtained by the proposed method spray drying of the composition according to the invention can be used in large economic scale in a wider range of products than a suspension, obtained directly �and stage iii).

As the carrier phase iv) is particularly preferable to use sodium sulfate. Preferably, the carrier amount, preferably sodium sulfate, is 4 times the amount of dry mass used suspension obtained in stage (iii). The total amount of dry matter used in stage v) mixture for spray drying is about 20 wt.%.

Spray drying according to stage (v) is preferably carried out with input temperatures used for spray drying gas input from 180 to 250°C and outlet temperature of the drying zone, usually a spray tower gas from 70 to 100°C., preferably 85°C, and the average stay is subject to drying of the material in the impact zone used for spray drying gas is from 10 to 60 seconds, preferably from 20 to 40 seconds. In other aspects, the spray drying specialist can refer to information application WO 01/25411 A1. This document describes in General suitable methods of spray drying to obtain dried by spray drying of enzyme products. Unexpectedly, it was found that when offered preferably high temperatures possible spray drying without significant loss of the ability of binding to S. mutans. This could be expected IP�odya from application WO 01/25411 A1, in particular, because the application does not contain a single example of a successful spray drying after pasteurization and, from the point of view of an expert, is speculative as to the suitability of spray drying, allegedly, for a very wide class of enzymes.

Therefore, especially preferred is the proposed method, according to which

(i) cells of one or more preferred strains of lactic acid bacteria L. paracasei or L. rhamnosus, preferably one or more of strains DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 and DSMZ 16673, particularly preferably at least DSMZ 16671, and/or the above-described mutant, or derivative cell line, in the above postexperimental the growth phase in the first stage is heated in suspension from the source temperature below 40°C to the temperature of pasteurization 78-80°C, preferably 80°C, with the change of temperature from 0.8 to 1.2°, preferably 1°C, after which

(ii) the suspension was kept for 25 to 35 minutes, preferably 30 minutes, at a temperature of pasteurization, and then

(iii) the suspension is cooled to a final temperature below 40°C with temperature variation from 0.8 to 1.2°, preferably 1°C, followed by washing with water as described above, by centrifugation and establish on the dry matter content of from 10 to 30 wt.%, preferably from 18 d� 22 wt.%, then

(iv) the suspension is mixed with a carrier to produce a mixture for spray drying, wherein the carrier is selected from sodium sulfate, potassium sulfate and calcium sulfate, and a mixture of two or more of them, and especially preferably represents a sodium sulfate, and wherein the amount of the carrier selected with providing the content of dry matter in the mixture for spray drying in the range of 10 to 30 wt.%, then

(v) obtained in stage (iv) suspension, equipped with a carrier, was spray dried at the temperature of the input gas for spray drying from 180 to 250°C and the temperature of the output gas from under the drying zone, usually drying tower, from 70 to 100°C., preferably 85°C, and the average time to be spray dried material in the impact zone of gas for spray drying is 10 to 60 seconds, preferably 20 to 40 seconds.

This method achieves the above advantages of the invention.

The proposed composition of lactic acid bacteria, capable of specific binding to Streptococcus mutans, received or obtained by the method comprising the stages (i) to (iii) and, if necessary, iv) and v), preferably administered in a product for use on the human body. When applied to the human body, in particular, the way� introduction into the oral cavity and/or of bringing it in contact with the teeth, you can in fact implement the prevention of dental caries using the present compositions.

It is advisable proposed articles have proposed a composition sufficient for binding to S. mutans and/or to achieve anticariogenic effect of the number. This number depends on the type of product, as well as its galenically form. In particular, the number depends on the desired measures of possible binding to S. mutans, respectively possible anticariogenic effect. By standard research specialist can easily determine the amount of the present composition, is sufficient depending on the desired effect, the type of product and its galenically form. In particular, the specialist will be aware that the product can be provided with a coating of the present composition or mix with this song.

Preferably, the product according to the invention refers to a group that includes food, taste food, beverages, prepared foods, tools for oral health, cosmetic and pharmaceutical products. The related products described in the application WO 2006/027265 A1.

Especially preferred products include chewing gum, toothpaste, rinses mouth and candy.

The invention is described below in more detail based on examples, and PR�measures do not limit the scope of protection of the claims.

Example 1: General method of obtaining

Cells of lactic acid bacteria, capable of specific binding to Streptococcus mutans and the specific binding is

(a) resistant to heat treatment and/or

b) resistant to treatment with protease and/or

in) dependent on calcium and/or

g) occurs in the range of pH between 4.5 and 8.5, and/or

d) occurs in the presence of saliva,

cultured in a suitable aqueous nutrient medium at a temperature of 37°C. Suitable nutrient medium contains glucose, yeast extract, Tween 80, and salt. The cultivation is carried out in the mode of recharge in the form of an aqueous suspension.

Once the content of glucose in the culture medium dropped below 1 mmol, or within 1 hour after this point, the slurry is heated to a pasteurization temperature of 75 to 85°C, preferably from 78 to 80°C. When heated slurry temperature change is from 0.5 to 1.2°C/min, preferably from 0.8 to 1.2°C/min.

The heated slurry was held at pasteurization temperature for 20 to 40 minutes, preferably 25 to 35 minutes, particularly preferably 30 minutes.

Then the suspension is cooled to a temperature below 40°C. At this temperature change is 0.5 to 2°C/min., preferably 0.8 to 1.2°C/min.

Thereafter, the cooled suspension by centrifugation con�antiroot to the dry matter content of 10 to 30 wt.%. The thus obtained concentrate was re-suspended in water and again concentrated by centrifugation to a dry matter content of 10 to 30 wt.%. Re-suspending and concentration is carried out in total up to 8 times. The result is a washed out a lot with the dry matter content of from 10 to 30 wt.%.

The resulting mass has the ability of specific binding to Streptococcus mutans. As such, you can enter in the food, taste the food, beverages, prepared foods, tools for oral health, cosmetic and pharmaceutical products, to give the corresponding product properties for the fight against tooth decay or enhance its existing properties to fight against tooth decay.

Example 2: a composition for the care of mouth

The culture of L. paracasei or L. rhamnosus, capable of specific binding to Streptococcus mutans by definition according to the invention, cultured, pasteurized and washed as in example 1. The pasteurization temperature is 80°C, the pasteurization time is 30 minutes. The temperature change every time is 0.8 to 1.2°C/min was Obtained after the first centrifugation of the concentrated suspension was re-suspended by adding water to the dry substance content of 5 wt.% in the newly obtained suspension. The thus obtained slurry was subjected�Ute re-centrifugation until the dry matter content of 20 wt.%.

To thus obtained is washed and concentrated mass is added aqueous media and fragrances. So get the tool mouthwash fit for the fight against tooth decay.

In the same way, get washed and concentrated masses from DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 and DSMZ 16673. And these masses are mixed with the aqueous medium and flavors to obtain the mouthwash to combat tooth decay.

Instead funds mouthwash get chewing gum, toothpaste and candy for the fight against tooth decay by entering the corresponding washed and concentrated mass in the bulk chewing gum, toothpaste and candy, and add the desired flavoring.

Example 3: General method, spray drying

Provide the basic solution for spray drying by obtaining water 10 to 30% solution of sulfate of an alkaline or alkaline earth metal. Obtained according to example 1, washed and concentrated mass re-suspended in four times the amount of the basic solution for spray drying for the formation of the mixture for spray drying, so that a volume of 100 mass units get 500 volume units of the mixture for spray drying.

The mixture for spray drying is subjected to spray drying by means of hearth�in the tower for spray drying. In the last injected gas for spray drying, which when fed to the tower for spray drying has a temperature of from 180 to 250°C, and with the release of the spray drying tower is from 70 to 100°C. the Average residence time of the mixture for spray drying tower for spray drying is not more than 60 seconds.

In the resulting spray-dried material retained the ability of specific binding to Streptococcus mutans described above in example 1. The resulting spray-dried material as such can enter into the food, taste the food, beverages, prepared foods, tools for oral health, cosmetic and pharmaceutical products, to give the corresponding product properties for the fight against tooth decay or gain in it existing properties to fight against tooth decay.

Example 4: further compositions for the care of mouth

The culture of L. paracasei or L. rhamnosus, capable of specific binding to Streptococcus mutans by definition according to the invention, cultured, pasteurized and washed as in example 1. The pasteurization temperature is 80°C, the pasteurization time is 30 minutes. The temperature change is from 0.8 to 1.2°C/min Obtained from the first centrifugation of the concentrated suspension was re-suspended patentabilty water to a solids content of 5 wt.% the newly obtained suspension. The thus obtained suspension was centrifuged again to the dry substance content of 20 wt.%.

Then the slurry was spray dried by the method described in example 3. The main solution for spray drying is a 20% aqueous solution of sodium sulfate. The inlet gas temperature to the spray dryer at the inlet to the tower for spray drying at 180-250°C. outlet temperature of the gas for spray drying with the release of the tower for spray drying is 70-100°C. the Average residence time of the mixture for spray drying tower for spray drying is 10 seconds.

Get dried by spray drying material, which is added to the aqueous media and flavors, resulting in getting the facility of mouthwash to combat tooth decay.

In the same way get dried by spray drying materials from DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672, respectively DSMZ 16673, and these materials are mixed with the aqueous medium and flavors to receive funds mouthwash to combat tooth decay.

Instead funds mouthwash you can get chewing gum, toothpaste and candy for the fight against tooth decay by introducing relevant material in the bulk for chewing gum, W�training of pasta or sweets and add the desired flavoring.

1. A method of producing compositions inanimate lactobacilli, capable of specific binding to Streptococcus mutans, comprising the following stages:
(i) heating a suspension of cells of a Lactobacillus or a mixture of lactobacilli, capable of specific binding to Streptococcus mutans and the specific binding is
(a) resistant to heat treatment and/or
b) resistant to treatment with protease and/or
in) dependent on calcium and/or
g) occurs in the range of pH between 4.5 and 8.5, and/or
d) occurs in the presence of saliva,
with the initial temperature below 40°C up to the pasteurization temperature of 75 to 85°C with temperature variation from 0.5 to 2°C/min,
ii) holding the heated slurry at pasteurization temperature for 20 to 40 minutes
iii) cooling the suspension with stage (ii) to a final temperature below 40°C with temperature variation from 0.5 to 2°C/min.

2. A method according to claim 1, characterized in that the cells of Lactobacillus selected from cells of Lactobacillus paracasei, Lactobacillus rhamnosus, or mixtures thereof.

3. A method according to claim 2, characterized in that the cells of Lactobacillus selected from cells of Lactobacillus paracasei or Lactobacillus rhamnosus, preferably with number DSMZ - DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 and DSMZ 16673, or a mutant or derivative cell lines, and in the mutant or derivative cell lines retained the ability of specific hydrogen bonds�ing with Streptococcus mutans.

4. A method according to claim 1, characterized in that the lactobacilli has the ability of specific binding to Streptococcus mutans serotype C (DSMZ 20523) and/or serotype e (NCTC 10923) and/or serotype f (NCTC 11060).

5. A method according to claim 1, characterized in that in stage (i) the pasteurization temperature is 78 to 80°C.

6. A method according to claim 1, characterized in that the change in temperature in the step (i) and/or (iii) is 0.8 to 1.2°C/min.

7. A method according to claim 1, characterized in that in stage (i) the suspension medium contains glucose in an amount of not more than 10 mmol /l.

8. Method according to one of claims.1-7, further comprising the following stages:
iv) mixing of step (iii) suspension with a carrier to produce a mixture for spray drying, wherein the carrier is selected from sulfates of alkali and alkaline earth metals, and wherein the amount of the carrier selected with the provision of the dry solids content of the slurry for spray drying from 10 to 30 wt.%,
v) spray drying of step (iv) mixture for spray drying.

9. A method according to claim 8, characterized in that the spray drying is carried out at inlet temperature used for spray drying gas from 180 to 250°C and the outlet temperature of the drying zone, usually a spray tower gas from 70 to 100°C, preferably 85°C, and the average time to be respiratorydisease material in the impact zone of gas for spray drying is 10 to 60 seconds, preferably 20 to 40 seconds.



 

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8 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Desulfovibrio sp. A 4/1 is deposited in the Russian Collection of Microorganisms (RCM) under the registration number of RCM B-2820D, and is resistant to heavy metal ions.

EFFECT: increase in degree of wastewater treatment.

2 tbl, 4 ex

FIELD: biotechnology.

SUBSTANCE: nutrient medium comprises enzyme-autolyzate of cattle spleen, monosubstituted potassium phosphate, disubstituted potassium phosphate, potassium gluconate, mesoinosite, soluble starch, gelatin, L-cysteine, iron (III) sodium salt, high-grade ink and distilled water in a predetermined ratio of components.

EFFECT: invention enables to improve the accuracy of determining of antibiotic susceptibility of cultures of legionellosis causative agent.

3 tbl 8 ex

FIELD: biotechnology.

SUBSTANCE: bacterium Bacillus subtilis is proposed, producing 5′-aminoimidazole-4-carboxamideriboside (AICAR). The said bacterium comprises deregulated pur-operon against the background of inactivated gene purH, the modified heterologous genes prs and purF E.coli under the control of a strong promoter PrpsF as part of chromosome. At that it has one of the following characteristics: comprises the gene zwf under control of a strong promoter PrpsF, comprises the heterologous gene udhA under the control of a strong promoter PrpsF, comprises the deletion of the gene sacB. The method of synthesizing AICAR by culturing the said bacteria under proper conditions is also proposed. At that culturing is carried out on the medium of the following composition, wt %: fodder yeast 0.5-1.0, sucrose 10-13, soy isolate 2.5-3.5, corn-steep extract 3.0-5.0, urea 0,6-0,8, (H34)23CO4 0.8-1.6, propinol 0.4-0.5, water - the rest.

EFFECT: high level of synthesis of AICAR.

5 cl, 2 tbl, 9 ex

FIELD: biotechnology.

SUBSTANCE: invention "Using bottom sea water of hydrogen sulphide pool of the Black Sea as sea algae culture medium" refers to marine culture. Technical substance of the invention consists in using bottom seawater of the Black sea both containing hydrogen sulphide, and oxidised as a sea algae culture medium. Studying the biogenic properties of the aqueous medium from a reduction zone of the Black sea implemented by the invention has shown that the bottom seawater has no bad influence on the Black Sea plankton algae in the presence of high initial concentrations of hydrogen sulphide. After complete oxidation of this xenobiotic, the Black Sea bottom water can be used as a nutrient fertile culture medium of unicellular plankton and multi-cellular benthoalgae in laboratory or industrial environment, and in marine culture farms.

EFFECT: sea algae culture in laboratory or industrial environment.

FIELD: food industry.

SUBSTANCE: invention relates to winemaking industry. "Quince -D" Saccharomyces cerevisiae yeast strain is deposited in the All-Russian Collection of Industrial Microorganisms (VKPM), Federal State Unitary Enterprise State Research Institute for Genetics, under Registration No Y-3973. Y-3973 strain has spore formation capability, ferments and digests glucose, sucrose, maltose, galactose, 1/3 of raffinose. Saccharomyces cerevisiae VKPM Y-3973 yeast strain has a high fermentative activity, well ferments quince wort containing sugar in an amount of 14.75 g/100 cm3 accumulating 8.79 % vol. of ethanol. The strain can more completely digest carbohydrates with formation of ethanol increased by 0.79 % vol. as compared to a conventionally known strain during the same period of fermentation. The specificity of the wine material produced with usage of Saccharomyces cerevisiae Y-3973 strain in a finer aroma and taste.

EFFECT: invention allows to produce high quality natural fruit-and-berry wines at the fermentation temperature equal to 20°C.

1 tbl, 1 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to biotechnology. Claimed is strain of Lactobacillus delbrueckii subspecies lactis CNCM I-3741, reducing content of cholesterol in blood. Strain is applied for obtaining fermented dairy products.

EFFECT: inventions provide efficient reduction of cholesterol content in blood.

3 cl, 3 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. The Arthrospira platensis (Nordst.) Geitl. rsemsu T/05-117 strain has high content of neutral lipids. The strain is stored in the collection of the research laboratory of renewable energy sources of the Faculty of Geography of Moscow Lomonosov State University.

EFFECT: invention increases output of neutral lipids.

6 ex

FIELD: biotechnologies.

SUBSTANCE: method of obtaining of brucellous L-antigen is performed as follows. The strain Brucella abortus 19 is cultivated in the L-form in the dense nutrient medium prepared on the basis of meat-peptonic hepatic glucose-glyceric agar (1.3-1.5%) with adding of 10-15% normal horse Serum at the temperature 20-22°C within 2-3 days. The culture, obtained in the L-form, is emulsified with 0.5% phenolised normal saline solution, inactivated at the temperature 85-90°C within 60 minutes. The suspension is centrifuged at 3000-5000 rpm within 15-20 minutes and the concentration of brucellas is established at the level 50-60 billion m.k. The obtained antigen is titrated, standardised by specificity and activity. It is used for agglutination test in serum diagnostics of brucellosis.

EFFECT: invention allows to minimise the time of antigen preparation and to increase the yield of bacterial mass.

4 tbl, 3 ex

Vortex bioreactor // 2538170

FIELD: machine building.

SUBSTANCE: vortex bioreactor includes cylindrical vessel1 with lid 2 provided with a device for medium mixing. Mixing device consists of bladed wheel 7, horizontally fixed on vertical shaft in upper part of vessel 1 and horizontal annular partition 9, installed in vessel 1 with a gap relative to its cylindrical walls, rod 10 vertically installed along axis. Horizontal annular partition 9 is located with possibility of rotation on vertical rod 10 and has retention mechanism of horizontal annular partition 9, installed on rod 10. Bioreactor is provided with pipe 19 or telescopic pipe 20, mated to axial hole 13 of horizontal annular partition 9, attached to the bottom of the last one and located around rod 10. On upper surface 11 of horizontal annular partition 9 there made are radial channels 12, located from axial hole 13 to the edge of annular partition 9 inclined downwards to the bottom of vessel 1. Horizontal annular partition 9 is designed floating, for example, from polypropylene. Inner diameter of pipe 19 or telescopic pipe 20 corresponds to diameter of axial hole 13 of horizontal annular partition 9. Area of axial hole 13 of horizontal annular partition 9 equals to cross sectional area at inlet 14 to radial channels 12 and total cross sectional area at outlet 15 from these radial channels 12.

EFFECT: improving efficiency of mixing and speeding up biochemical processes upon their carrying out using fluid mediums of various viscosity.

6 cl, 5 dwg

FIELD: biotechnology.

SUBSTANCE: banana fruit and skin are weighed, washed, crushed and mixed with water preheated to 45±1°C in the ratio of 1:3. It is alkalised to pH 8.2-8.3, the enzyme is added, which is used as the pancreas of cattle in the amount of 9-15% of total volume. The preservative is added, which is used as chloroform in an amount of 1% of total volume. The mixture is subjected to hydrolysis for 10 days at a temperature of 45-50°C. During the first 24 hours the mixture is stirred every 15 minutes for 5 minutes, and then every 2 hours for 5 minutes. The hydrolyzate is filtered through cheesecloth, then the filter towels (paper) and sterilised by ozonation. It is covered with cork and stored hermetically sealed at a temperature of from 2°C to 8°C.

EFFECT: invention provides obtaining of transparent hydrolyzate with increased sterility, with high biological activity, which leads to improvement of its properties when it is used as a growth stimulator of Leptospira.

2 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: method provides culturing in the liquid culture medium of the virulent strain of bacteria Staphylococcus aureus No. 6 followed by separation of the culture medium from the bacteria by filtration through the filter PLASMAFILTER PLASMAFLUX PSu 2S with obtaining the filtrate. Ammonium sulphate to 80% saturation is added to the resulting filtrate to obtain the precipitate. The resulting precipitate is separated by centrifugation at 10000 g for 30 minutes and dissolved in phosphate buffer at pH 7.4 with subsequent microfiltration of the protein-containing fraction through the 0.22 mcm membrane and desalting on the column PD-10, purification and concentration by carrying out ion-exchange chromatography on the column of Q-sepharose, elution with 0.15 M NaCl and ultrafiltration on two filters of 100 and 30 kDa to obtain protein containing fraction with the molecular weight of 30-90 kDa and protein content of 0.5-1.0 mg/ml.

EFFECT: invention enables to obtain the products based on protective secreted protein-containing compound.

1 dwg, 5 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method to cultivate sublimated strains of microorganisms includes introduction of a growth stimulant and a source of carbon into a dense nutrient medium with subsequent seeding of microorganism cells, incubation of seeds and accounting of viable microbial cells. Sublimated strains of microorganisms include Yersinia pestis EV, Escherichia coli C600, Bacillus anthracis "СТИ", Vibrio cholerae NAG 504, Brucella abortus 19BA. The growth stimulant and carbon source is 96% ethyl alcohol in the amount of 1.0-2.0% of the medium volume. If necessary, they add gentian violet in concentration of 1:100000.

EFFECT: improved yield of viable strain cells on dense nutrient media.

1 tbl, 5 ex

FIELD: biotechnology.

SUBSTANCE: two suspensions are prepared. Clinical polyantibiotic-resistant strains Escherichia coli are added to an isotonic solution of NaCl to achieve the concentration of 30-40 thousand CFU/ml. Copper nanoparticles are added to the solution of NaCl to achieve the concentration of 0.01-0.05 mg/ml. The suspension of ethylenediaminetetraacetic acid - EDTA is prepared by its dilution in distilled water at the rate of 0.1-0.2:1, respectively. NaOH is added to the prepared suspension to obtain the solution of EDTA with pH=7.6-8. The prepared suspensions are connected with the solution of EDTA in the following ratios by wt %: suspension of copper nanoparticles - 70-85, suspension of microorganisms - 10-20, EDTA - 5.10. It is incubated in the shaker at 100-150 rev/min and a temperature of 36-38°C for 40-60 minutes. The resulting biomass is inoculated on the solid nutrient medium with the volume of 20-25 ml in the amount of 0.1-0.12 ml. It is incubated in the thermostat at a temperature of 36-38°C for 18-24 hours. The sensitivity of E.coli strains to antibiotics is determined.

EFFECT: invention enables to increase the sensitivity of the said bacterial strains to antibiotics gentamicin and ampicillin.

2 tbl, 2 cl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, namely to leukolectins, and can be used in medicine. What is prepared is the polypeptide leukolectin characterised by SEQ ID NO:1-8. The recombinant preparation is ensured by using a nucleic acid coding it and integrated into an expression vector which is used to transform a host cell. Testing absence-presence or determining an amount of the polypeptide leukolectin are ensured by using an antibody or an antigen-binding fragment of a variable region of the above antibody which is specifically bound to the polypeptide leukolectin. The polypeptide leukolectin or the nucleic acid coding it are used as ingredients of a pharmaceutical composition in therapy of pathological disorders of skin and mucous membranes.

EFFECT: invention enables treating or preventing autoimmune disorders of skin, inflammatory diseases of skin or mucous membrane, or injured skin in an animal effectively.

16 cl, 19 dwg, 3 tbl, 12 ex

FIELD: ecology.

SUBSTANCE: soil contaminated with oil products is detoxified by adding a natural sorbent with a biopreparation until a given concentration of the contaminant in the soil is achieved. The sorbent used is glauconite and the biopreparation used is polyculture. Before adding the sorbent with the biopreparation, concentration of the contaminant is measured, the volume of sorbent is determined and a shielding intermediate layer of biohumus is placed at the boundary where the contaminant penetrates the soil in the lower part of the contaminated layer. Concentration of the contaminant is determined layer-by-layer. The number of layers of contaminated soil with concentration of the contaminant in the layer differing from each other by at least double is not less than two. The contaminated soil is moistened after distributing the volume of sorbent with the biopreparation, effective microorganisms and fermented compost on the surface of the contaminated soil while simultaneously mixing. A water-swelling polymer is also added to the contaminated soil. Temperature conditions for activating biological processes are maintained by adding organic materials to the soil, wherein the volume of said organic materials in the shielding intermediate layer should be at least double that in the contaminated soil.

EFFECT: high efficiency and faster detoxification.

1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and can be used for methane-producing cell permeability control. What is prepared is a polypeptide able to permeate into a methane-producing cell and to increase its permeability, characterised by an amino acid sequence SEQ ID NO:117, 118 or 119 or being at least 90% identical to the above sequence, or at least 15 sequential amino acids of the above sequence. What is also prepared is a polynucleotide coding the above polypeptide cloning and expressing vectors used for producing host cells producing the polypeptide or used for the vector replication. The polypeptide can contain a fluorescent tag on an N-terminal amino acid residue.

EFFECT: invention enables providing higher methane-producing cell permeability.

18 cl, 35 dwg, 3 ex

FIELD: agriculture.

SUBSTANCE: invention relates to biotechnology, in particular to methods of breeding beneficial insects. The method comprises preparing the nutrient medium comprising soy flour, sucrose, milk powder, palm kernel oil, dry aphides, Wesson salt, dry brewer's yeast, tocopherol, ascorbic acid, agar-agar, vitamins B1, B6, B12, metaben, inositol, and distilled water at a predetermined ratio and growing coccinellidae Harmonia axyridis Hall. at a temperature of 23-26°C in the prepared medium, applied to pieces of sterile polyethylene film. At that the change of feed is carried out daily.

EFFECT: invention enables to simplify breeding coccinellidae Harmonia axyridis Hall.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to cosmetic industry and represents a cosmetic product which has the following composition in each specific case calculated using the total composition: at least 0.1 wt % of at least one hydrophilic softening product, 2 to 40 wt % of at least one surfactant specified in a group of fatty alcohol ethoxylates, fatty alcohol ether sulphates and salts of sulphated and/or sulphonated fatty acids, 30 to 90 wt % of water, 1 to 30 wt % of one or more abrasives with the total ingredients making 10%; the product contains at least 0.1 wt % of at least one hydrophilic softening product with a hydrophilic-lipophilic balance ≥8 and flour thermally treated by saturated vapour; the flour is natural flour of shell or kernels characterized by a light absorption at wave length 660 nm of less than 1 prepared by reacting the flour 1 g with a solution prepared of water 10 ml and 0.1% aqueous methylene blue 1 ml.

EFFECT: invention provides the lower effect on viscosity, possesses the high cleaning action and high tolerability.

16 cl, 5 ex, 3 tbl

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