Method of plant material fermentation and bacteria cultivation, extract of fermented plant material, powder of fermented plant material extract, and their application

FIELD: medicine.

SUBSTANCE: method involves preparation of nutrient medium including plant material, plant material fermentation with anaerobic gram-negative bacterium with simultaneous bacterium cultivation in the medium to obtain extracts of fermented plant material. Extraction is performed by water or salt buffer. Also the extract can be optionally dried to obtain extract powder. Edible plant material containing carbohydrates, mainly polysaccharides, is used as plant material. Gram-negative bacterium lives only in symbiosis with given plant material. Additionally invention claims application of extracts and powder of fermented plant material in obtainment of medicines, veterinary medicines, quasi medicines, cosmetics, food products, including functional food, forage and detergents.

EFFECT: obtaining products with immunostimulating effect.

17 cl, 1 dwg, 23 tbl, 23 ex

 

The technical field to which the invention relates

The present invention relates to a method of fermenting and culturing to obtain immunostimulant, which is safe for addition to drugs, drugs for animals, quasilocality, cosmetics, foods, functional foods, food products and detergents for mammals, including humans (especially domestic animals, Pets and the like), birds (especially grown Chicks, poultry and the like), amphibians, reptiles, fish (especially farmed fish, pet fish, etc. and invertebrates, the method of obtaining the extract of the fermented plant extract of fermented plants containing immune obtained by the method fermentation and cultivation, the powder containing the immunostimulant derived from an extract of fermented plants, and the composition of the fermented extract of a plant containing the extract of fermented plants.

Prior art

The actual problem is the prevention of disease and methods of treatment, including prevention of infection in mammals, including humans (especially domestic animals, Pets and the like), birds (especially grown Chicks, poultry and the like), AMF is Biy, reptiles, fish (especially farmed fish, pet fish, etc. and invertebrates. Moreover, to achieve this much-needed ways without the use of chemical reagents, without pollution, without obtaining resistant bacteria and accumulation in the human body. The authors of the present invention have found for the above problems that Immunostimulants vegetable origin, such as an aqueous extract of wheat, reliably prevent the disease and have a therapeutic effect (patent document 1, non-patent document 1). To achieve the above effect, the authors of the present invention found that it is possible to use low molecular weight lipopolysaccharide derived from Pantoea agglomerans, which is a symbiotic bacterium for wheat (non-patent document 2). While recent studies have shown that various substances in addition to the lipopolysaccharides are immunomoduliruushim (immunostimulating effect, and that many natural materials containing immune, attracted attention.

Technology fermentation using microorganisms is generally used not only in food industry but also in broader areas. Fermentation has been widely applied to obtain the JV the mouths including wine, obtain from soy sauces and pastes from beans, soy products, fermented milk, such as cheese, and to obtain drugs. There are many microorganisms that are used for these types of fermentation, and examples are yeast rice malt (fungus) and lactic bacteria, but rare cases of application of the gram-negative bacteria. In the main fermentation is a phenomenon in which organic matter is decomposed under the action of the microorganism, and includes, in a broad sense, the formation of a microorganism useful substances (non-patent document 3). Examples of the use of microorganisms in fermentation include winemaking. Winemaking is a technology fermentation using adhesion of the yeast to the grape skins and its product is alcohol. The technology of fermentation using a microorganism known to use such as gram-negative bacteria, fermentation methane using methane bacteria, acetic fermentation of acetic fungi and fermentation ethanol fermentation of tequila) from rhizomes American agave, usingZymomonas mobilisbut hardly known fermentation culture using edible plants as material and microorganism, characterized by a symbiotic interaction with the plant, and the p immunostimulant never attracted attention as a product of fermentation. Furthermore, the method of fermentation and cultivation with the aim of obtaining immunostimulant never attracted attention.

Thus, when performing fermentation using a microorganism, basically, there are conditions of nutrition for growth of the microorganism, which fermentary substrate must meet. That is, the presence of substances consumed by microorganisms as nutrients, it is necessary, for example as sources of carbon are contained in sufficient quantities monosaccharides, such as glucose and fructose. Therefore, fruits such as rich in fructose grapes, can be used as a substrate fermentation without carrying out any processing. However, in other cases, the required pre-processing, such as heating and processing of the enzyme for the fermentation of this organism. For example, the aboveZymomonas mobilisis a microorganism used for fermentation of tequila. In this case, the polysaccharides obtained from the rhizomes of the American agave, which is not an edible plant, transform into formatiruem monosaccharides by heating, and then the sugars are fermented by the microorganism with obtaining alcohol as a product of fermentation. Therefore, when you create a culture fermentation using conventional microorganism is a, polysaccharides, such as starch, is not acceptable as a substrate fermentation. For example, described the fact thatPantoea agglomeranscan't break down the starch (non-patent document 4).

The inventors have shown that the active component for potentiation of immunity contained in the aqueous extract of wheat flour (non-patent document 5). The inventors have also shown that the active ingredients are contained in food grains (wheat, rice), seaweed (brown algae, kelp, hijiki (brown alga) and red algae) and beans (soy beans and bean paste) (non-patent document 6). With regard to biological activity, it was found that these objects have a preventive effect against diseases of humans and mice (diabetes, hyperlipidemia, atopic dermatitis, cancer) and can be effective in the prevention of infections in fish, shellfish and chicken (patent document 1, non-patent document 1). However, for the above expectations of the effect of aqueous extract of wheat flour necessary to take into wheat flour in a large number.

ThisPantoea agglomeransis a bacterium which lives in a symbiotic interaction with wheat, and is believed to be applicable in the cultivation of wheat, because the bacteria provides wheat phosphorus and nitrogen (non-patent document 7. AlsoPantoea agglomeransis deposited not only in wheat but also in the epidermis of pears and apples. In Europe it has been shown that disease decay caused by fungi can be prevented at the time of sedimentation of the bacteria, and priority was the development of the use of this bacterium as an environmentally friendly non-toxic fungicide (non-patent document 8). It was determined that the symbiosis is "a phenomenon in which alien organisms live together. In this case, traditionally refers to maintaining behavioral or physiological proximity. Therefore, under this concept misses accommodation only in the same habitat. Symbiosis is classified and divided into different categories depending on the meaning of life and the importance of the symbiotic partner, sustainability, interaction and spatial distribution of the symbiotic partner. Basically, symbiosis in a broad sense is divided into three types, mutualism, kommensalizmom and parasitism on the basis of the presence or absence of the benefit/harm of the symbiotic partner (non-patent document 9). It is known thatPantoea agglomeransseparated from the wheat in any regions and for any types of (non-patent document 5) and also separated from the fruit (non-patent documents 10, 11). It was shown thatPantoea agglomeransprotects plants is of fungi or other bacteria through the production of antibiotics (non-patent documents 12, 13) and provides fixation of phosphorus and nitrogen (non-patent document 7). Therefore, I believe thatPantoea agglomeransalways present in plants and plays a role beneficial to the plants. Thus, the nature of its activity is defined as "symbiosis", but not "parasitism". In addition, the authors showed that the active component for potentiation of immunity contained inPantoea agglomerans. Also, the authors found that low molecular weight lipopolysaccharide obtained from this bacteria, has a preventive effect against disease in humans and mice (diabetes, hyperlipidemia, atopic dermatitis, cancer) and effective for the prevention of infection in fish, shellfish and chicken (patent document 3, non-patent document 2).

In this case, the authors imbued with the idea of creating a method of obtaining an extract of fermented plants, usingPantoea agglomeransas a method of obtaining safe and affordable immunostimulant. That is, the inventors have focused on (1) inexpensive cultivationPantoea agglomeransusing a medium containing major protein components included in the solution for cultivation, derived from plants, as well as component, a fermentation plant, and (2) receipt of materials enriched byPantoea agglomeranscontained in the plant or the product f is mentaly, thus, getting drugs, drugs for animals, quasilocality, cosmetics, functional foods, food products, feed products and detergents for mammals, including humans (especially domestic animals, Pets and the like), birds (especially grown Chicks, poultry and the like), amphibians, reptiles, fish (especially farmed fish, pet fish, etc. and invertebrates. However, this does not mean that the microorganism living in symbiotic interactions with plants, can directly use the vegetable components, for example the material of edible plants as substrate for fermentation. For example, wheat flour is a complex organic substance consisting of starch and similar substances present in the grains wheat, but a dedicatedPantoea agglomeransthat is symbiotic microorganism of the outer shell of the wheat, not to have direct contact. Thus, with the help of symbiotic interaction between wheat and microorganism cannot show whetherPantoea agglomeransto ferment wheat to be cultivated using wheat or not. Indeed, unknown and not published data on the ability ofPantoea agglomeransto assimilate wheat flour. On the contrary, on the basis of well-known facts would be the about described, whatPantoea agglomeranscan't use wheat starch as substrate for fermentation.

Glucide contained in plants, often stored in the form of starch, and it is noteworthy for edible plants, particularly food grains. Typically, the microorganisms do not have the function in which the starch is assimilated to a high degree. In this regard, it became known that some facultative gram-negative bacteria can ferment starch. For example, it is known thatErwiniaable to assimilate the starch. However, in this fermentation, when fermented starch, refers to the use of amylase activity of the microorganism by adding a microorganism cultured in large quantities in different optimal environment, and was never intended that the culture itself is created using starch and therefore is fermentation. In conventional technology, this technology meets the objective fermentation only for the effective use of amylase activity of the microorganism and is not planned to grow the microorganism using starch as substrate. In the examples of the present invention describes what fermentary product is produced in addition to the cultivation of the microorganism, using starch as ed is stannage source of carbon, and the present invention differs significantly from conventional technology, the fact that this example is not only the fermentation, but also fermentation and cultivation.

On the other hand, if a particular microorganism retains its function decomposition of starch, it does not directly mean that the microorganism can grow, using as substrate starch. At the end of cultivation, in the case that the growth of a microbe, the number of the added microorganism is extremely small. In this case, even if the organism is poorly expressed amylase activity, this activity is too low for sufficient decomposition of the substrate and the growth of the microorganism is not achieved. Indeed, it was found that many microorganisms cannot grow, using starch as the sole carbon source.

However, if the fermentation and cultivation can be performed using thePantoea agglomeransin a medium containing wheat flour as a main component for the formation of a fermented extract of the plant (hereinafter extract of fermented plants, obtained by fermentation and cultivation ofPantoea agglomeransin a medium containing wheat flour as a main component, corresponds to the extract of fermented wheat)containing b is lsom number budget immunostimulant, as specific examples should be provided with medicines, drugs for animals, quasilocality, cosmetics, foods, functional foods, food products and detergents that do not pollute the environment, is safe and effective for the prevention of infection in humans and in animal production and aquatic crops. The present invention was accomplished through the use of the discovery thatPantoea agglomeransgrew up using wheat flour as substrate in the above context and due to the intensive conducting many experiments.

The term extract of fermented plants introduced by the present invention, is generic, which includes the actual culture solution obtained by the implementation of the fermentation and cultivation, the liquid component obtained by separating solids/liquids of this culture solution, and a liquid component obtained by the impact of the extraction process on a solid component obtained by the separation of solids/liquids, and so forth. Thus, the extract of fermented plant includes a culture solution obtained by the method of fermentation and cultivation in accordance with the present invention, and all extracts, which can be obtained IP is the use of all or part of the culture solution. Although, of course, it goes without saying that the extract of fermented plants can be processed by drying, as in the case of a powder extract of fermented plants, or by dissolving the extract of fermented plants with optimum concentration in a suitable solution such as phosphate buffer solution, including normal saline.

[patent document 1] Japanese unexamined patent application No. H3-218466

[patent document 2] Japanese unexamined patent application No. N8-198902

[patent document 3] WO 00/57719

[patent document 4] Japanese unexamined patent application No. N6-78756

[patent document 5] Japanese unexamined patent application No. N4-187640

[patent document 6] Japanese unexamined patent application No. N4-49240

[patent document 7] Japanese unexamined patent application No. N4-99481

[patent document 8] Japanese unexamined patent application No. H5-155778

[non-patent document 1] Inagawa, H. et al., Biotherapy 5(4), p617-621, 1991

[non-patent document 2] Soma, G. et al., "Tumor necrosis Factor: Molecular and Cellular Biology and Clinical Relevance p203-220, 1993

[non-patent document 3] Yamada, T. et al., "Seibutsugaku Jiten" 3rded., p1021, 1983

[non-patent document 4] Gavini, F. et al., Int. J. Syst. Bacteriol., 39), p337-345, 1989

[non-patent document 5] Nishizawa, T. et al., Chem. Pharm. Bull., 40(2), p479-483, 1992

[repatent the second document 6] Inagawa, H. et al., Chem. Pharm. Bull., 40(4), p994-997, 1992

[non-patent document 7] A. H. Neilson, J. Appl. Bacteriol., 46(3), p483-491, 1979

[non-patent document 8] Nunes, C. et al., Int. J. Food Environ., 70(1-2), p53-61, 2001

[non-patent document 9] Yamada, T. et al., "Seibutsugaku Jiten" 3rded., p287-288, 1983

[non-patent document 10] Nunes, C. et al., J. Appl. Environ., 92(2), p247-255, 2002

[non-patent document 11] Asis C. A. Jr. et al., Lett. Appl. Environ., 38(1), p19-23, 2004

[non-patent document 12] J. L. Vanneste et al., J. Bacteriol., 174(9), p2785-2796, 1992

[non-patent document 13] Kearnes L. P. et al., Appl. Environ. Environ., 64(5), p1837-1844, 1998

Description of the invention

The problem solved by this invention

As has been described, Immunostimulants often contained in the plants and are often components or products of microorganisms, which live in symbiotic interactions with plants. Therefore, to obtain the immunostimulant of natural product, safe at reception, it is necessary to extract this component directly from edible plants (e.g., limulus-positive glycolipid, patent document 1) or effectively to cultivate the microorganism that lives in a symbiotic interaction with edible plant, for its component or product (for example, low molecular weight lipopolysaccharides. Patent document 2). However, the content immunostimulant in edible plant very mA is about, a very large amount of food must be digested, so you can expect an immunostimulating effect in the consumption of food, and, basically, it is not easy to keep the quantity of consumed immunostimulant in the appropriate form. Thus, one should not expect its impact. Moreover, extraction immunostimulant from the plant and use it as food or drugs is associated with high costs and has low practical significance.

When focusing on the organisms that live in a symbiotic interaction with the plant,Pantoea agglomeransthat is symbiotic bacterium wheat, contains as a component of low molecular weight lipopolysaccharide, effective for immunostimulation. To date, however, for the extraction of low molecular weight lipopolysaccharide was necessary to cultivatePantoea agglomeransusing expensive environment in which the main protein found in the environment is of animal origin, such as NZ amine, tripton or casamino acid. Therefore, it was difficult to inexpensively provide a highly available immunostimulant. At the same time, cannot be rejected for the unknown probability of harmful substances, for example, those found in animals infected with BSE in cattle (BSE).

A means of addressing this problem

The method of fermentation and cultivation of the present invention, which includes the fermentation of the material obtained from edible plants and containing glucide of which the main component is a polysaccharide, facultative anaerobic gram-negative bacterium, which lives in a symbiotic interaction with the plant, and the simultaneous cultivation of anaerobic gram-negative bacteria.

Fermentation and cultivation can be carried out in a simple way the fermentation of starch as a carbon source with facultative anaerobic gram-negative bacteria.

Preferably, facultative anaerobic gram-negative bacterium was a facultative anaerobic Bacillus.

Preferably, facultative anaerobic Bacillus belonged to the familyEnterobacteraceae.

Preferably, the optional is anaerobic Bacillus belonged to the genus Pantoea,SerratiaorEnterobacter.

Using facultative anaerobic BacillusPantoea agglomeransmay make starch carbon source.

It is also desirable that the edible plant was selected from cereal grains, seaweed, beans, and mixtures thereof.

It is also desirable that the edible plant was a food grain and material of food grains selected from wheat flour, rice powder, powder wheat bran, rice bran and sediment from the sake. In particular, since wheat flour contains gluten as a protein source, there is the opportunity to ferment and to cultivate even without the use of material of animal origin.

Preferably, the edible plant was seaweed and material derived from sea algae selected from the powder brown seaweed, powder "mekabu (sporophyllUndariacrategusand kelp powder.

When edible plants are beans and material derived from soy beans, are a waste fermenting the beans, this material contains large amounts of protein. Thus, there is the ability to efficiently ferment and cultivate even without the use of material of animal origin.

The fermented extract of a plant of the present invention is produced by way of fermentation and cultivated the I.

Powder of fermented extract of a plant of the present invention is obtained from the extract of fermented plants.

The composition of the fermented extract of a plant of the present invention contains an extract of fermented plant or powder of the extract of fermented plants.

The composition of the extract of fermented plants can be selected from drugs, drugs for animals, quasilocal, cosmetics, food, functional food products, feed products and detergents.

Preferably, the fermented extract of the plant had the following physico-chemical properties.

The fermented extract of the plant shows the ability to activate macrophages even in the presence of polymyxin b C. the fermented Extract of the plant has immunostimulating effect.

Preferably, facultative anaerobic gram-negative bacterium was a Bacillus that belongs to the genusPantoeaand edible plant was selected from cereal grains, seaweed, beans, and mixtures thereof.

Preferably, facultative anaerobic gram-negative bacterium was aPantoea agglomeransand edible plant was selected from cereal grains, seaweed, beans, and mixtures thereof.

It is desirable that the material obtained is C cereal grains, chose from wheat flour, rice powder, powder wheat bran, rice bran and sediment from sake.

It is desirable that the material derived from seaweed, was chosen from a powder brown seaweed mekabu powder and kelp powder.

The effect of the invention

In accordance with the present invention due to the fact that cultivation is carried out in an environment that does not contain animal ingredients, no contamination by impurities of animal origin. Therefore, there is no possibility of contamination unknown harmful substances, for example, derived from BSE-carriers, and are able to create highly secure and inexpensive method of obtaining an extract of fermented plants that can meet the needs of different assignments and safely and inexpensively to provide a fermented extract of a plant or a powder of fermented extract of a plant containing the immunostimulant. Moreover, it is possible to provide culturally solution, immunostimulant, extract and powder of the extract and additional drugs, drugs for animals, quasilocality, cosmetics, foods, functional foods, food products and detergents containing extract or powder of the extract.

Never p who was ideologues and there are no facts research traditional fermentation technology, regarding the fact that fermentation and cultivation can be carried out in a simple way, so that the material derived from edible plants, fermented facultative anaerobic gram-negative bacterium, which lives in a symbiotic interaction with the plant and simultaneously cultivated facultative anaerobic gram-negative bacterium.

This can be used if TNF is produced by macrophages or no induction activity of TNF) as an indicator that the substance exhibits an immunostimulating effect. Moreover, immune-stimulating effect can be quantified by measuring the amount of produced TNF. Thus, the production of TNF by macrophages was measured using the limulus-positive plant glycolipid derived fromPantoea agglomerans. Production of TNF by macrophages was stopped by treatment with polymyxin b and in the case of limulus-positive plant glycolipid derived from wheat flour, and in the case of low molecular weight lipopolysaccharide derived fromPantoea agglomerans. However, in many examples it was shown that even when the extract of fermented plants were treated with polymyxin B, TNF was producyrovtsa macrophages. This shows that the extract of fermented plant derived EN zymes is the situation and cultivation, has immunostimulating effect, qualitatively different from the immunostimulatory effects of the components of the plant, which was material, and the microorganism used for fermentation.

Brief description of drawing

The drawing is a drawing showing the inhibiting effect of the presence of koi herpes through feed product containing the extract of fermented wheat.

The best mode of applying the invention

The respective embodiments of the present invention will be described in detail below.

I. the Essential feature extract fermented plants is the use ofPantoea agglomerans.

In the present invention by the authors was discovered for the first time, thatPantoea agglomeranscan grow directly using as a carbon source starch, and usingPantoea agglomeranswas created the way for inexpensive extract of fermented wheat abundantly containing as a product of fermentation and product cultivation immunostimulant. This method can provide an environmentally friendly and safe quasilocality, cosmetics, food, functional food and feed products that are effective for the prevention of infections in humans and livestock, and aquatic culture.

1. SelectionPantoea agglomeans

When wheat flour is suspended in water and the supernatant is applied on L-broth agar medium and cultured appear colonies of microorganisms. In these colonies of microorganisms are identified by standard methods. For example, those that have properties identical with the standardPantoea agglomeranschoose a selection of colonies that are painted as gram-negative, positive reactions of anaerobic glucose metabolism and negative in relation oxidase activity and when using ID-test (Nissui Pharmaceutical Co., Ltd). StandardPantoea agglomeransavailable from the Institute of physical and chemical research, Bioresource center (non-patent document 4). In the following description the percentage is the value by weight, unless otherwise specified.

2. Evaluation of immunostimulatory activity

In these embodiments in quality indicator immunostimulating effect, which manifests extract of fermented wheat were evaluated for their ability activation of macrophages by measuring TNF production by macrophages.

3. Low molecular weight lipopolysaccharide derived fromPantoea agglomerans

With regard to one of the active components, immunostimulatory fermentation and cultivation usingPantoea agglomerans, it was expected that it contains low molecular weight lipopolysaccharide, polucen the th of Pantoea agglomerans. Low molecular weight lipopolysaccharides are particularly pronounced security and exceptional biological activity compared to the commonly used high-molecular lipopolysaccharides (normal lipopolysaccharides). Thus, the measured content of low molecular weight lipopolysaccharide. Low molecular weight lipopolysaccharide described in patent document 2. These examples relate to the extract of fermented wheat, but the present invention does not imply that the plant is limited to wheat and that used immunostimulant is limited by the low molecular weight lipopolysaccharides.

Pantoea agglomeranscan be cultivated and using well-known methods (patent document 2, non-patent document 8), but the basic components of proteins contained in the culture medium, are of animal origin, and value of the environment is high. Moreover, when the animal give functional food, functional feed products or injected subcutaneously, contamination by impurities of animal origin, it is relevant to BSE, is the issue of food safety, and in addition, the production becomes expensive and the method is of no practical significance. Thus, as a result of extended studies is to get a harmless and inexpensive natural product, having immunostimulatory effects, the authors present invention has created a way of fermentation and cultivation usingPantoea agglomeransto obtain the extract of fermented wheat, as shown in the examples. The main component of the protein contained in the culture medium, was usually animal protein, as in the present invention is a protein of vegetable origin. Normally, the product obtained by the decomposition of protein, such as casein, derived from cow's milk using a digestive enzyme, was added into the culture solution. In this case, the cost of a liter of medium was 250 yen, and the use of wheat flour cost is approximately 16 yen. Never fermentation was carried out with the aim of significant concentration and sinergeticheskogo merge immunostimulating activity of plants and microorganisms in their symbiotic interaction.

The contents of this invention will be described below by way of examples, but the present invention is not limited to the use ofPantoea agglomeransas the microorganism, wheat, as edible plants, or flour, as the material described in these examples. The present invention can also be applied to the material obtained in the usual way from other edible plants, Abel is about containing immune, for example, brown algae, cereal grains (material obtained from cereal grain is wheat flour, rice powder, powder wheat bran, rice bran or sludge from sake), seaweed (material derived from seaweed, is a powder seaweed mekabu powder or powder kelp) and beans (material derived from soy beans, are a waste fermented beans). It is well known that these plants contain proteins and sugar. These plants can be used for fermentation and cultivation with the use ofPantoea agglomerans. It is well known that natural bacteria, for example, bacteria belonging to the genusSerratiaorEnterobacterlive in symbiotic interaction with these plants (non-patent document 4). Needless to say that the microorganisms used for the fermentation, include facultative anaerobic gram-negative bacteria, which live in a symbiotic interaction with these plants.

II. A brief summary of the main points of the present invention

(1) Extract of fermented wheat itself as a substance that has immunostimulating effect is created by combining wheat,Pantoea agglomeransthat is symbiotic bacteria, and fermentation products, their combination is new, but this is General the invention is not limited to this only.

(2) what is New is the obtaining of an extract of fermented plants usingPantoea agglomeranswhich is a gram-negative bacterium, but the present invention is not limited to this only.

III. A specific method of obtaining the extract of fermented wheat

(1)Pantoea agglomeransextracted from wheat flour standard method (non-patent document 1). Once isolated and identified, this bacterium can be stored in 50% glycerol.

(2) are 0.05-5% salt solution, 0.005 to 1 molar phosphate buffer, or a mixed salt solution (0.5-10% solution of sodium phosphate, 0.05 to 5% solution of potassium dihydrophosphate, 0.05 to 5% solution of sodium chloride, 0.05 to 5% solution of ammonium chloride).

(3) Wheat flour suspended in water at a concentration of 0.05-10%.

(4) Prepare 0.2 to 3 molar solution of magnesium chloride.

(5) Prepare 0.2 to 3 molar solution of calcium chloride.

(6) the Solutions to (2) to (5), in some cases, sterilized by autoclaving, etc.

(7) the Solutions of (2) through (5) are mixed in an appropriate amount and add water to create a suspension containing 0.1-5% wheat flour. In some cases, the pH is neutralized by adding a solution of alkali or acid.

(8) In some cases, wheat starch can be partially cleaved by the addition of from 10 to 50,000 units of amylase per liter of medium in (7) and incubation at a temperature of 10 is about 80°C for 1 to 24 hours.

(9)Pantoea agglomeransselected in (1), add (7) or (8).

(10) (9) fermented at a temperature of from 1°to 40 ° C. In some cases, fermentation tank can simply stand or be stirred up. Alternatively, mixing may be carried out every few hours.

(11) (10) fermented for from 6 hours to one week. As the fermentation solution of wheat flour turns yellow.

(12) Optional can be added to the alkaline solution to neutralize the pH during fermentation (11) or can be added to the slurry of wheat flour or inorganic salt.

(13) the Fermentation is stopped, and the solid component is collected as a precipitate by centrifugation (1000-5000 rpm, 10-60 minutes). Sludge can be used directly as the product of fermentation of wheat flour for food products or as raw material for mixing with the feed material.

(14) In the case of obtaining an extract of fermented wheat (13) is suspended in water or buffered saline, which is then heated at a temperature of from 80 to 140°C for from 10 minutes to 6 hours. The solid component can be removed by centrifugation or filtration. Water or buffer can be added again to the remote draught for re-extraction with heating for several times.

(15) Extract the farm is mounted wheat, obtained in (14)can be further simply cleaned, depending on the anticipated needs. Thus, a precipitate is formed when a salt such as sodium chloride, at a final concentration of 0.05-1 mol/l is added to the extract (14), and further add the solvent, such as ethanol, from one to three times the number compared to the number of the extract. The precipitate can be collected by centrifugation. The precipitate may be further washed with a solvent, such as ethanol. When drying the precipitate can be obtained powder.

A. EXAMPLES RELATING TO the METHOD of OBTAINING the EXTRACT of FERMENTED WHEAT

Example 1

Study of the growth ofPantoea agglomeransin a medium containing wheat flour

To confirm whether thePantoea agglomeransnatural symbiotic bacteria to wheat can grow using wheat flour as the source of carbon was estimated growthPantoea agglomeransin a medium containing wheat flour.

(1) Preparing M9 agar medium containing 0.5% of wheat flour as carbon source.

(2) a Single colony ofPantoea agglomeranswere taken from the surface of LB agar medium and suspended in 1 ml phosphate buffer. It was further diluted in 10-10000 times, and 0.1 ml of each aliquot were sown on M9 agar medium (1).

(3) After cultivation at 37°C for 6 days was observed the presence of colonies. Ka is the result, watched about 300 colonies in the Petri dish, which was sown 0.1 ml 10000-fold dilution.

This confirms that thePantoea agglomeranscan use wheat flour as a carbon source.

Example 2

Obtaining an extract of fermented wheat

(1) Distilled water (5 ml) was added to 0.5 g of wheat flour for the suspension, 0.1 ml of supernatant was added to L-broth agar medium and cultured at 37°C during the night.

(2) Yellow colonies were isolated, the bacteria were identified by standard methods, was isolated byPantoea agglomerans, suspended in 50% glycerin solution and kept in the freezer. Part of the original substance was applied onto LB-agar medium, were left at 37°C for independent colonyPantoea agglomerans.

(3) a 2-liter flask was added 64 g of hydrogen phosphate sodium heptahydrate, 15 g of potassium dihydrophosphate, 2.5 g of sodium chloride, and 5 g of ammonium chloride, and purified water to obtain a total volume of 1 liter (mixed solution of inorganic salts). Purified water was added to 13.1 g of dihydrate magnesium chloride to obtain a total volume of 100 ml (solution of magnesium chloride). Purified water was added 11.1 g of calcium chloride to obtain a total volume of 100 ml (solution of calcium chloride). Purified water (4 l) was added to a 5-liter conical flask (cleaned the bedroom water). The above solutions and purified water sterilized by autoclaving (TOMY BS-325, 120°C for 20 minutes).

(4) Wheat flour (24 g) (Nisshin Flour Milling Co., Ltd.) added to 1-liter conical flask and was added purified water to obtain a total volume of 600 ml, After the same autoclave was added 3 mg of α-amylase (SIGMA, Bacillus, enzymatic activity of 1500-3000 units per mg protein), and was heated on a water bath at 65°C for 12 hours (solution of wheat flour treated with amylase).

(5) the Obtained solutions and the like in amounts shown in table 1, were placed in a 3-liter sterilized Sakaguchi flask to obtain a medium containing wheat flour.

Table 1
MaterialsDose
A mixed solution of inorganic salts200 ml
Purified water550 ml
The solution of wheat flour treated with amylase200 ml
The solution of magnesium chloride2.0 ml
The solution of calcium chloride0.1 m the

(6) Preparation of inoculum: a Single colony ofPantoea agglomeransisolated from wheat flour (2), was added to 10 ml of medium containing wheat flour (5), previously prepared with the same composition, and fermentatively with weak stirring at 37°C overnight (12-15 hours) to obtain the inoculum for the fermentation of wheat flour.

(7) All number (6) was added to (5) and fermentatively at 37°C for 20-30 hours with stirring. Measured pH fermentive solution and brought to pH 7 by addition of an aqueous ammonia solution. In sterile 150 ml of wheat flour was treated with amylase and he was added to 37.5 ml of a mixed solution of inorganic salts and similarly was fermentatively 20-30 hours. The next step was repeated to achieve a total fermentation time 65-80 hours.

(8) the Solution of the fermented wheat flour was centrifuged (Hitachi, high speed refrigerated centrifuge, SCR-20B, 5000 rpm, 20 min, 4°C) and the precipitate was collected.

(9) To the precipitate (8) was added to phosphate buffer, which is then added to obtain a total volume of 100 ml per aliquot in 33 ml was transferred into a 50-ml centrifuge tube, and extraction was performed with heating in a boiling water bath for 30 minutes. After heating, the solution was cooled to room temperature and centrifuge holds Aravali (Hitachi, high speed refrigerated centrifuge, SCR-20B, 10,000 rpm, 20 minutes, 20°C). After centrifugation-decantation another test tube was collected 82 ml of supernatant pale yellow color.

(10) a Solution of sodium chloride (8,9 ml, 5 mol) was added to 80 ml of the supernatant in (9). When the solution was added 178 ml of ethanol, there was a white suspension. The solution was left in a freezer (-90°C) overnight and then the solution was centrifuged (Hitachi, high speed refrigerated centrifuge, SCR-20B, 10,000 rpm, 20 min, 4°C). The precipitate was obtained by removal of the supernatant. After adding to the precipitate with 10 ml 70% ethanol and subsequent suspension solution was centrifuged (Hitachi, high speed refrigerated centrifuge, SCR-20B, 10,000 rpm, 20 minutes, 20°C.) and the precipitate washed. The precipitate was dried in air and dissolved in the distilled water with 11 ml of the extract of fermented wheat.

(11) Measurement of dry mass: 0.3 ml was transferred into a pre-weighed 1.5 ml plastic tube, and after freezing using lyophilizate was conducted by lyophilization, and the mass came to 7.45 mg Therefore, the dry weight of the extract of fermented wheat (10) amounted to $ 24.8 mg / 1 ml solution and 273 mg on the total number of 11 ml.

(12) Extract of fermented wheat was 8 times independently in the same way, and the amount of protein in each about what ASCE was measured by the method of Bradford, using as a standard protein, bovine serum albumin (BSA). As objects of comparison were used limulus-positive glycolipid (patent document 1) and low molecular weight lipopolysaccharide (patent document 2). The results of the measurements are presented in table 2. In tables 2-5 and 7, the numeric value for the extract of fermented wheat represented as the content of mg to 1 g mass obtained by drying the extract of fermented wheat obtained in the above (10).

(13) Measurement of sugar: the sugar content was estimated using persulfate method, using glucose as the standard sugar. The measurement results are shown in table 3.

(14) the Measurement of nucleic acids: to evaluate the absorption of the sample diluted 100 times at a wavelength of 210-340 nm. The maximum content was calculated using a value obtained by subtracting the absorption at 320 nm from the absorption at 260 nm and 50 µg per unit of optical density of DNA absorption. The measurement results are shown in table 4.

(15) Measurement of the content of limulus-active substances using the limulus-analysis: measure was used Toxi-color system Seikagaku Corporation and Seikagaku Corporation Et-1 as a standard limulus-active substances. The measurement results are shown in table 5.

(16) Iodobromide reaction: 1 h iodine reagent (10 ml of water was added 12.7 g of iodine and 25 g of potassium iodide, thoroughly mixed, and then added water to 100 ml) was diluted 200 times with water when used. This solution (5 ml) was added to 0.1 ml of the extract of fermented wheat, pre-diluted to obtain a concentration of 1 mg/ml, and were thoroughly stirred. In the solution of the extract of fermented wheat immediately developed color from light purple to dark purple (positive reaction). In limulus-positive glycolipid and low molecular weight lipopolysaccharide this procedure is not induced staining (negative reaction). The above results are summarized in table 6.

As is evident from the above results, the extract of fermented wheat differs from limulus-positive glycolipid and low molecular weight lipopolysaccharide in protein, sugars, nucleic acids (except limulus-positive glycolipid, because for him there is no data on the content of the limulus-positive substances and iodobromide reaction, and it is clear that the present substance is new. The above results are summarized in table 7. Thus, the extract of fermented wheat in these examples is new and different from limulus-positive glycolipid and low molecular weight lipopolysaccharide that is showing the following physico-chemical properties. Extract the farm is mounted wheat shows the protein content of 5-15%, the sugar content of 20-45%, the content of nucleic acids 10-35% and the content of the limulus-positive substances 10-40% and positive in iodobromide reaction and is capable of activating macrophages even in the presence of polymyxin b C.

Table 2
The protein content in fermented extract
SampleThe protein content (mg/g)
Extract of fermented wheat 160
Extract of fermented wheat 271
Extract of fermented wheat 390
Extract of fermented wheat 4105
Extract of fermented wheat 5103
Extract of fermented wheat 682
Extract of fermented wheat 788
Extract of fermented wheat 888
limulus-positive glycolipid40
Low molecular weight lipopolysaccharide3.8 or less

Table 3
The sugar content in fermented extract
SampleThe sugar content (mg/g)
Extract of fermented wheat 1318
Extract of fermented wheat 2428
Extract of fermented wheat 3313
Extract of fermented wheat 4232
Extract of fermented wheat 5372
Extract of fermented wheat 6324
Extract of fermented wheat 7298
Extract of fermented wheat 8329
limulus-positive g is taliped 133
Low molecular weight lipopolysaccharide668

Table 4
The content of nucleic acids in fermented extract
SampleThe content of nucleic acids (mg/g)
Extract of fermented wheat 1102
Extract of fermented wheat 2102
Extract of fermented wheat 3226
Extract of fermented wheat 4291
Extract of fermented wheat 5302
Extract of fermented wheat 6240
Extract of fermented wheat 7218
Extract of fermented wheat 8216
limulus-positive glycolipid Not published
Low molecular weight lipopolysaccharide2,8

Table 5
The content of limulus-active substances in fermented extract
SampleThe content of limulus-active substance (mg/g)
Extract of fermented wheat 1242
Extract of fermented wheat 2118
Extract of fermented wheat 3125
Extract of fermented wheat 4458
Extract of fermented wheat 5224
Extract of fermented wheat 6231
Extract of fermented wheat 7356
Extract of fermented wheat 8289
limulus-positive glycolipid 970
Low molecular weight lipopolysaccharide993

Table 6
Iodo-starch reaction fermented extract
SampleDefinition
Extract of fermented wheat 1Positive
Extract of fermented wheat 2Positive
Extract of fermented wheat 3Positive
Extract of fermented wheat 4Positive
Extract of fermented wheat 5Positive
Extract of fermented wheat 6Positive
Extract of fermented wheat 7Positive
Extract of fermented wheat 8Positive
limulus-positive glycolipidNegative
Low molecular weight lipopolysaccharideNegative

Example 3

Immunostimulatory effect of the extract of fermented wheat

Cell line acute myelogenous leukemia, TPR-1 (1×106/250 µl RPMI1640 medium containing 10% fetal calf serum), used as human macrophages were placed in a 48-well plate and pre-cultured for 30 minutes. Next, 250 μl of medium (final volume 500 μl) was added so that the final concentration of each sample was 1-10000 ng/ml of the samples were added polymyxin B (12.5 µg/ml). After culturing for 4 hours collecting cultural supernatant and cells. The activity of TNF in the supernatant was measured cytotoxicity test using L-929. The results are shown in table 8. Macrophages were produced TNF even in the presence of polymyxin b by the action of the extract of fermented wheat, but low molecular weight lipopolysaccharide and limulus-positive glycolipid in the presence of polymyxin b could not affect the synthesis of TNF by macrophages. On this basis, it is evident that the extract of fermented wheat has a biological asset is awn, other than those that have low molecular weight lipopolysaccharide and limulus-positive glycolipid.

Century example of the USE of the EXTRACT of FERMENTED WHEAT FORAGE PRODUCTS

Example 4

Feedstuff for growing chickens, containing extract of fermented wheat (effect of inhibition mortality in rearing broilers in a large-scale study)

Produced feedstuff containing 430 mg/kg of the extract of fermented wheat obtained in example 2. Commercial broiler chickens were used in a number of 5500-6000 Chicks in the group. In the control test group was given feed product containing the extract of fermented wheat. Feed product containing the extract of fermented wheat gave the chickens at the age of 3 weeks after hatching and introduced every day until the age of 7 weeks. The number of dead Chicks were counted every day. Chicks that did not meet the standard during transportation, wypracowanie. The results are shown in table 9. The rate of removal in the test group was 1.9% (feed product containing the extract of fermented wheat), which was low, and 3.3% in the control group. The survival rates in the test group amounted to 98.1% and 96.7% in the control group. Thus the m there was an increase in survival rates, equal to 1.4%. Was carried out test significant differences in the number actually carried chickens and the number of remote chickens between test group and control group, was observed a significant difference p<0.0001 in X2test. Based on the above, it was shown that the effect of protection from infection due to the feed product containing the extract of fermented wheat in growing broilers.

Table 9
Effects of feed product containing the extract of fermented wheat growing broilers
The test groupThe control group
The number of Chicks59065525
The number actually carried Chicks57925345
The number of remote(culled) Chicks114180
The level of discarding1,9%3,3%
98,1%96,7%

Example 5

Feed product for farmed fish, containing the extract of fermented wheat (the effect of preventing infection in yellowtail, test outdoors)

To evaluate the effect of preventing infection test outdoors approximately 5200 of gelthveau per group were fed with feedstuff containing extract of fermented wheat obtained in example 2. The results are shown in table 10. The mortality rate due toStreptococcusin the control group was 4.8%. In the group that received 100 mg/kg/day (body weight of 1 kg and one day) (test group), mortality was observed significantly decreased (p<0,00001) compared to the group not taking the specified feed product (control group).

Table 10
Prevention of infection in yellowtail due to the action of the feed product containing the extract of fermented wheat test outdoors
ImpactThe number of surviving fishThe number of dead fishTest significant differences (X2test)
The control group52012494,79
The test group51931011,94(P<0,00001)

Example 6

Feed product for farmed fish, containing the extract of fermented wheat (the effect of preventing infection koi herpes)

(1) Carp: we investigated black carp weighing 70, the Test was performed in a group containing 20 Karpov.

(2) Obtaining virus koi herpes: added 10 ml of a balanced buffered salt solution Hank to 1 g of the bronchi carp, who died from infection koi herpes, and homogenized, filtered through a filter with pore diameter of 0.45 μm, and the filtrate was a solution of the virus.

(3) virus Infection koi herpes: the above filtrate (600 μl/100 g body weight) was injected intraperitoneally.

(4) Preparation of feed product containing the extract of fermented wheat extracts of fermented wheat obtained in example 2 was mixed with commercially available food product at a concentration of 0, 5, 10 and 20 mg/kg

(5 Method of feeding: each feedstuff in the amount of 1% of body weight given once a day. This corresponds to 0, 50, 100 or 200 µg/kg body weight/day in terms of the number of extract of fermented wheat.

(6) Experiment: Feed product containing the extract of fermented wheat gave during the week, then carp infected with a virus, and then feed product containing the extract of fermented wheat was given for 10 days. Observed survival rates of carp within 10 days after virus infection. The result is shown in the drawing.

All carp to the end of the sixth day died in the group that were not given the extract of fermented wheat. It was shown that in the groups that were given the extract of fermented wheat, the level of survival was significantly higher on day 10 after infection (method of Kaplan-Meier, log rank criterion, the percentage of risk amounted to 0.01% or less). In particular, the level of survival is equal to 65%, was shown in the group, which was given 100 mg/kg body weight/day of the extract of fermented wheat.

C. APPLICATION EXAMPLES extract of FERMENTED WHEAT IN COSMETICS AND IN OBTAINING DETERGENTS

Example 7

Getting hand cream containing extract of fermented wheat

For more ointment about 10% extract of fermented wheat obtained in example 2 was mixed with ointment ironist Oronogo substrate 1 in the composition, described in table 11.

Table 11
TrackDose
White petrolatum250 g
Stearyl alcohol200 g
Propylene alcohol120 g
Castor oil 60, utverjdenie a polyoxyethylene40 g
Glycerol monostearate10 g
Methylparahydroxybenzoate1 g
Propylparabens1 g
Purified waterThe required number

Example 8

Getting a moisturizing cream containing extract of fermented wheat

1. Preparation of a moisturizing cream containing extract of fermented wheat

Components are presented in table 12. Combination And were heated and dissolved at 70°C, the combination was mixed in purified water in an amount of 1/4 and heated/dissolved at 70°C, and the combination With eremetical in purified water in an amount of 1/4 and heated/dissolved at 70°C, and added in combination A. the Mixture was thoroughly stirred by a homogenizer and then cooled to 40°C. Then was added a combination of D and the pH was brought to 6.8. Then added the remaining purified water and extract of fermented wheat obtained in example 2 in an appropriate amount, and thoroughly stirred to obtain milk. Extract of fermented wheat pre-dissolved in purified water to a concentration of 5 mg/ml and 0.1 ml was added to 100 g of milk.

Table 12
ComponentsWt./wt.%Combination
Squalene5,0And
Olive oil10,0And
Jojoba oil5,0And
Stearic acid4,0And
The monostearate polyoxyethylenesorbitan (EO)1,8And
Methylpolysiloxanes 0,3And
The monostearate sorbitan0,5In
Glycerol monostearate savemarriage type3,0In
Propylparabens0,2In
Methylparahydroxybenzoate0,2In
1,3-Butyleneglycol5,0In
Concentrated glycerin6,0In
Carboxyquinolone0,22
The potassium hydroxideThe required numberD
Extract of fermented wheat (5 mg/ml)0,1
Purified waterThe required number
The total number of 100,0

2. Moisturizing cream containing extract of fermented wheat

This cream was used 43 men and women, and was conducted by questionnaire. As a result, regarding moisturizing effect, 18 people answered that he had a moisturizing effect, 18 people answered that took place a light moisturizing effect, 2 people replied that it was not moisturizing effect, and 5 people did not respond (criterion mark one sample: p<0,0001). Regarding the effect of improving the action on the rough skin of 6 people answered that the cream was definitely effective, 13 people answered that the cream was slightly effective, no one said that the treatment was ineffective, and 24 people did not respond (criterion mark one sample: p<0,0001). Regarding the deterioration of the skin after applying the cream nobody said deterioration. This cream has been used by 4 people with moderate atopic symptoms, and was conducted by questionnaire. Regarding the improvement of atopic dermatitis 3 people answered that the cream was definitely effective, and one person said that the cream was slightly effective (criterion mark one sample: p<0,125). In addition, one person said that the scars from acne quickly passed. This cream was used with 9 men after shave,and was conducted by questionnaire. Eight of the men said that the cream was effective in reducing pain after shaving, preventing dryness and fast healing of cuts (criterion mark one sample: p<0,01). In addition, this cream was used by 2 people with age-related symptoms "frozen shoulder" by drawing on the shoulder to reduce the pain. One person said that the cream was effective.

In addition, this cream has been used by patients with burns. In patients with equally burn injury on the skin of both hands, one hand put the cream containing extract of fermented wheat, and on the other hand - cream, not containing extract of fermented wheat. Hand treated with cream containing extract of fermented wheat, obviously healed faster. This cream was used in 10 patients with burn injuries, including the described case. Therefore, in all places, treated with cream containing extract of fermented wheat, wounds healed faster than in areas treated with cream containing extract of fermented wheat (exact convergence in probability Fischer: p<0,0001). From the above, it was shown that the extract of fermented wheat showed a therapeutic effect against burn injuries.

Example 9

Obtaining skin lotion containing the extract of fermented wheat

1. Preparation of a skin lotion containing the extract of fermented wheat

The components used are shown in table 13. Extract of fermented wheat obtained in example 2 was pre-dissolved to obtain a concentration of 5 mg/ml in purified water, and 0.1 ml was added to 100 g of skin lotion.

Table 13
Components%
Sodium citrate0,1
The sodium pyrrolidone carboxylate1,0
1,3-Butyleneglycol5,0
POE(30) pop(6) decyltetradeceth ether0,6
Purified waterThe required number
Extract of fermented wheat (5 mg/ml)0,1
PreservativeThe required number
Ethanol10,0
The total number of100,0

2. Effective what s skin lotion, contains extract of fermented wheat

This skin lotion used 5 women, and was conducted by questionnaire. As a result, 3 women said that they felt good moisturizing effect and 2 women replied that he felt normal moisturizing effect. None of the women had no skin problems.

Example 10

Obtaining a detergent containing extract of fermented wheat

Detergent containing extract of fermented wheat was obtained to improve body functions. The main components of the detergent are shown in table 14.

Table 14
ComponentsContent
The sodium sulfate25,0 g
The calcium silicate0.26 g
Odorant (yuzu (citrus junos))0.5 g

Detergent containing extract of fermented wheat, were prepared by adding 110 g of extract of fermented wheat obtained in example 2, to the above components. Detergent containing extract of fermented wheat and washing medium is in, not containing extract of fermented wheat arbitrarily gave 102 subjects, which are then used them in the bath (160-200 liters) before taking a bath, and was conducted by questionnaire [(1) the degree of heating of the body, (2) feeling cold after a bath, (3) the effect of fatigue, (4) ease of falling asleep, (5) the degree of removal of the feeling of "frozen shoulder", (6) the effect of muscle pain, (7) effect of nerve pain, (8) the effect of lower back pain, (9) the effect of sensitivity to low temperatures, (10) the effect of improvement of ringworm of the foot (11) the effect of improving the condition of dry skin (12) effect against atopic dermatitis]. As a result, observed 7% or more improvement compared with the control (1) the degree of heating of the body (10%), (2) the complexity of feeling cold after a bath (7,9%), (6) the effect of muscle pain (13%), (8) the effect of low back pain (16%), (9) the effect of sensitivity to low temperatures (10%) and (11) the effect of improving the condition of dry skin (7,3%) (test Mantel-Haenszel: p<0,04). From the above results was observed effects in relation to pain and improve heating body in the application of the extract of fermented wheat as a detergent.

D. APPLICATION EXAMPLE extract of FERMENTED WHEAT FORAGE PRODUCTS

Example 11

Getting candy containing extract of fermented wheat

(1) as the original Mat is rials mixed granulated sugar, starch syrup, a mixture of water and extract of fermented wheat obtained in example 2 in a ratio of 5:5:5:1 and was prepared by heating from 120 to 160°C.

(2) Candy was obtained by cooling the mixture obtained in (1), in steel tanks for cooling, stretching to obtain the form of sticks and forming grains by weight, about 1 year

These candies in an appropriate amount were placed in 20 ml of water and dissolved by heating. The number of lipopolysaccharide, the active component of the extract of fermented wheat was measured in this solution, and, therefore, the content was 4.6 ág/g, These candies were eaten 6 men and women who have caught a cold and had a sore throat. This was followed by a questionnaire about pain in the throat. About sore throat all 6 people felt less pain in the throat (criterion mark one sample: p<0.03 in).

Example 12

Obtaining a functional product, destructive alcohol containing extract of fermented wheat

Extract of fermented wheat obtained in example 2 was mixed with a commercially available product, functional product that destroys the alcohol, and was evaluated, it was observed whether a new action, expressed in facilitating pharyngodynia or not.

Commercially available product: trade mark "Nonde oiki"

Components of a CR is dstanley in table 15.

Table 15
ComponentsThe content of the component
Powdered sugar78,98%
Vitamin C10,00%
Toyride-Pto 5.00%
Vitamin B20,02%
Odorant(menthol)0,50%
Amachazuru (Gynostemmapentaphylla) (saponin)3,50%
T-Flavor Conc 13189B (flavonoids)2,00%

This "Nonde oiki" contains extract machazuru (Gynostemma pentaphyllaand green tea extract, but only in the amount of, approximately, of 0.002 micrograms per serving of lipopolysaccharide, which is one of the active components of the extract of the plant. Therefore, it is expected that the product will have a new effect by adding the appropriate quantity of extract of fermented wheat, which abundantly contains lipopolysaccharide. It is desirable to combine from 1 to 30 μg to 2 g portion of lipopolysaccharide, which is one of the active compound is an extract of fermented wheat (from 5 to 150 μg in the case of the extract of fermented wheat). Thus, the first was produced product, which is combined with 50 μg of extract of fermented wheat in one portion. In the production process Nonde oiki was added 2.5 mg of the extract of fermented wheat per 100 g of product. The result has been a new product that contained 50 μg of extract of fermented wheat on 2 g of the product.

Analyzing 20 adult men and women who complained of pharyngodynia after drinking and singing karaoke, 10 people were given traditional "Nonde oiki" and "Nonde oiki", containing the extract of fermented wheat, respectively, and were evaluated by the increasing destruction of alcohol, which is well-known, and the effect of facilitating pharyngodynia. Immediately after that was conducted a questionnaire survey about the effect of easing pharyngodynia. As a result, decreased pharyngodynia in 8 out of 10 people who took "Nonde oiki", containing the extract of fermented wheat, against 2 people who took the traditional "Nonde oiki". Thus, there was a statistically significant difference (exact convergence in probability Fischer: p<0,0001) compared to control.

That is, the EXAMPLE CONCERNING the MEDICAL USE of EXTRACT of FERMENTED WHEAT

Example 13

Obtaining glycerin solution containing the extract of fermented wheat (therapist the economic effect against atopic dermatitis)

50%glycerol solution containing 50 μg/ml of extract of fermented wheat obtained in example 2 gave 2 or 3 times per day with a dosage of 2 to 3 ml per one introduction 9 patients, male and female with stable atopic dermatitis (age 25 to 34), where there has been a rash on the face, hands, feet, all over the body, neck, arms and back and subjective symptoms were moderate to severe. Subjective symptoms (itching) were classified into weak, moderate and expressed on the basis of patient complaints. After a period ranging from two weeks to two months after the start of application of patients visited again and the effects were evaluated. As a result, the cases of complete response (significant improvement of the rash and the almost complete disappearance of subjective symptoms) was 4 (44%), partial response (slight improvement of the rash and reduce subjective symptoms) was 4 (44%), non-response were equal to one (11%) and degradation was not (criterion mark one sample: p<0.03 in). From the above result, the level of effectiveness was defined as equal to 89%.

Example 14

Analgesic effect of the extract of fermented wheat

Extract of fermented wheat obtained in example 2, was dissolved in distilled water and 0.2 ml was administered to mice orally using the m probe. After 90 minutes, the mice were injected intraperitoneally of 0.7% acetic acid. After observing the mice for 5 minutes counted the number of vigibase caused within 30 minutes. The results are shown in table 16 as the number of each sample required for inhibition of 30% in the number of vigibase, when used as a control of distilled water. When the effective action of low molecular weight lipopolysaccharide fromEscherichia coliwas equal to 1, the effective action of the extract of fermented wheat was equal to 7, demonstrating that the extract of fermented wheat had an excellent analgesic effect.

Table 16
Analgesic effect of the extract of fermented wheat on pain induced by acetic acid in mice
ImpactThe number of induction 30%inhibitionRelative activity
Distilled water230±190 mg1
Extract of fermented wheat33±35 mg7,0

Example 15/p>

The inhibitory effect of the extract of fermented wheat in relation to atopic dermatitis

To assess the effect of the extract of fermented wheat in relation to atopic dermatitis was introduced model allergies 1st type. Intravenously injected antigenicity-monoclonal antibodies (1 μg/mouse) to male mice of BALB/c mice (3-4 mice per group). After one hour intradermally injected extract of fermented wheat obtained in example 2 (abdominal region) (4 µg/mouse), or oral (100 μg/mouse). After an additional hour, 20 μl of a solution of acetone and olive oil, mixed in the ratio (4:1)containing 0.25% dinitrofluorobenzene, was applied as an allergen on the surface and the back side of the ear of a mouse. The thickness of the ear was measured using calibrater after 1, 2, 24 and 48 hours after application. The value of (∆)obtained by subtracting the thickness prior to application, was the degree of swelling. The effect of drug administration was assessed using the level of inhibition obtained using the following formula in inhibiting the early stages of the reaction, the observed one hour after the introduction of the allergen, and delayed responses that are induced after 24 hours. The level of inhibition = (1-∆ swelling of the auricle after drug administration/∆ swelling of the auricle in control×100). R. the results are presented in table 17. As it follows from the table, the extract of fermented wheat inhibited allergic reaction and intradermal and oral introduction.

Table 17
The inhibitory effect of the extract of fermented wheat in relation to allergic reactions
The route of administration of extract of fermented wheatDosage (/mouse)The level of inhibition (%) (after one hour)The level of inhibition (%) (after 24 hours)
Intradermal injection4 mcg81,0102,1
Oral administration100 mcg41,360,8

Example 16

The effect of preventing contamination of the extract of fermented wheat

To evaluate the effect of preventing contamination of the extract of fermented wheat was introduced infectious model of methicillin-resistantStaphylococcus aureus(MRSA). Intraperitoneally injected cyclophosphamide (CY, 200 mg/kg) to male mice of BALB/c (at the age of 68 weeks) (10 per group), and after 5 days were injected intradermally extract of fermented wheat obtained in example 2. After 3 hours intravenously injected MRSA (3×107colony forming units (CFU)) and assessed the number of days of survival. The results are presented in table 18. As can be seen from the table, the extract of fermented wheat showed the effect of preventing infection with the statistically significant difference test (X2: p<0,001) compared with the control group treated with saline solution.

Table 18
The prophylactic effect of the extract of fermented wheat against infection MRSA
The injectable preparationThe survival ratesRisk level
Saline0/10
Extract of fermented wheat (0,004 g)9/10p<0,001
Extract of fermented wheat (0.04 µg)6/10p<0,005

Example 17

therapeutic effect of the extract is ArmeniaNow wheat in relation metastasises cancer

To assess therapeutic effect of the extract of fermented wheat against metastatic cancer was introduced model of metastasis in lung cell line Meth A. Intravenously injected cancer cells Meth A (1×105cells) male mice of BALB/c mice (aged 6-8 weeks) (10 per group) and after 12 days were injected intradermally for 4 days in a row, the extract of fermented wheat obtained in example 2. Twenty days after transplantation of the cells was performed the autopsy, lung were removed and were fixed with formalin. Light was observed by the naked eye and counted the number of nodes. The results are presented in table 19. As can be seen from the table, the extract of fermented wheat showed a therapeutic effect against metastatic lung cancer Meth with A statistically significant difference (t-test: p<0,001) compared with the control group treated with saline solution.

Table 19
therapeutic effect of the extract of fermented wheat in relation metastasises lung cancer A Meth
The injectable preparationThe number of nodes (value±standard deviation)Risk level
Saline60±11
Extract of fermented wheat (40 µg/kg)33±8p<0,001
Extract of fermented wheat (400 µg/kg)19±6p<0,001

F. EXAMPLES relevant extract of the WASTE FERMENTED BEANS

Example 18

Obtaining extract of the waste fermented beans

(1) 1.0 l of water, 0.2 g of potassium dihydrophosphate and 1.15 g of sodium hydrogen phosphate, 8 g of common salt and 0.2 g of potassium chloride were added to a 2-liter conical flask.

(2) the Dried waste fermentation of beans (20 g) was added to (1).

(3) (2) sterilized by autoclaving.

(4) Preparation of inoculum: a single colony ofPantoea agglomeransisolated from wheat flour was added to 5 ml of 2%environment waste fermenting the beans, pre-cooked with the same composition, and parenterally at 37°C overnight (15 hours) with careful stirring to prepare the inoculum for the fermentation of waste fermenting the beans.

(5) the Total number (4) was added to (3) and fermentatively at 37°C for 48 hours accurate mixing.

(6) the Solution of the extract of the waste fermented beans (5) was extracted by heating at 120°C for 20 minutes in autocl the E. Centrifuged (Kubota 8800, 2000 rpm, 10 minutes) and the supernatant was collected to obtain an extract of the waste fermentation of beans.

(7) Determination of dry weight: 0.3 ml was transferred into a 1.5 ml plastic tube, weighted, and after freezing the lyophilization was performed using lyophilizate, and then the weight was 5,97 mg. Therefore, the dry weight of the extract of the waste fermented beans (6) amounted to 19.9 mg / 1 ml solution and 19.9 g total 1000 ml.

(8) the Amount of protein was determined in the sample, diluted 10 times using the method of Bradford using BSA as a standard protein. The results are presented in table 15.

(9) the Determination of nucleic acids was determined by absorption at a wavelength of 210-340 nm sample, diluted 100 times. The maximum content was determined using a value obtained by subtracting the absorption at 320 nm from the absorption at 260 nm and 50 µg per unit of optical density of the absorption of DNA.

(10) Determination of sugar: the sugar content was determined by phenol-sulfate method, using as the standard sugar glucose.

(11) Determination of limulus-active substances limulus-analysis: to determine the system was used Toh-color provided by Seikagaku Corporation, and Seikagaku Corporation Et-1 was used as the standard limulus-active substances. The result is presented in table 20.

Table 20
The content of components in the extract of the waste fermented beans
Components(mg/g)
Protein112
Sugar537
Nucleic acidNot defined
Limulus-active substance10

Example 19

Immunostimulating effect of the extract of the waste fermented beans

Cell line acute myelogenous leukemia TNR-1 (1×106/250 μl of RPMI1640 medium containing 10% fetal calf serum), used as human macrophages were placed in a 48-well plate and pre-cultured for 30 minutes. Next, 250 μl of medium (final volume 500 μl) was added so that the final concentration of each sample was 100-10000 ng/ml, the Samples were supplied by the group containing polymyxin B (12.5 ág/ml) (not group containing polymyxin In only at 100 ng/ml). After culturing for 4 hours collected supernatant cultures and cells. The activity of TNF in which supernatant was measured using the test of cytotoxicity, using L-929. The results are presented in table 21. Macrophages were produced TNF even in the presence of polymyxin b under the influence of the extract of the waste fermentation of beans, but in the presence of polymyxin b, macrophages were not able to produce TNF under the influence of low molecular weight lipopolysaccharide. From this it is evident that the extract of the waste fermentation of beans has a biological activity that is different from the one that has a low molecular weight lipopolysaccharide.

G. examples of the extract of FERMENTED RICE POWDER

Example 20

Obtaining an extract of fermented rice powder

(1) 1.0 l of water, 0.2 g of potassium dihydrophosphate and 1.15 g of sodium hydrogen phosphate, 8 g of common salt and 0.2 g of potassium chloride were added to a 2-liter conical flask.

(2) Dried rice powder (20 g) was added to (1).

(3) (2) sterilized by autoclaving.

(4) Preparation of inoculum: a single colony ofPantoea agglomeransisolated from wheat flour was added to 5 ml of 2%medium rice powder, pre-prepared with the same composition, and parenterally at 37°C overnight (15 hours) with careful stirring to prepare the inoculum for the fermentation of rice powder.

(5) the Total number (4) was added to (3) and fermentatively at 37°C for 72 hours accurate mixing.

(6) Solution of fermented rice powder (5) was extracted by heating at 120°C for 20 minutes in an autoclave. Centrifuged (Kubota 8800, 2000 rpm, 10 minutes) and the supernatant was collected to obtain an extract of fermented rice powder.

(7) Determination of the content of limulus-active substances limulus-analysis: to determine the system was used Toh-color provided by Seikagaku Corporation, and Seikagaku Corporation Et-1 was used as the standard limulus-active substances. The content of limulus-active substances in the extract of fermented rice powder was 1.7 µg/ml.

Example 21

Immunostimulating effect of the extract of fermented rice powder

Cell line acute myelogenous leukemia TNR-1 (1×106/250 μl of RPMI1640 medium containing 10% fetal calf serum), used as human macrophages were placed in a 48-well plate and pre-cultured for 30 minutes. Next, 250 μl of medium (final volume 500 μl) was added so that the final concentration of each sample was 100-10000 ng/ml, the Samples were supplied by the group containing polymyxin B (12.5 µg/ml). After culturing for 4 hours collected supernatant cultures and cells. The activity of TNF in supernatants was measured using a cytotoxicity test using L-929. The results are presented in table 22. Macrophages were produced TNF even in the presence of polymyxin b under the influence of the extract Fe is matirovannogo rice powder, but in the presence of polymyxin b In macrophages could not produce TNF under the influence of low molecular weight lipopolysaccharide. From this it is evident that the extract of fermented rice powder has a biological activity, other than those that have low molecular weight lipopolysaccharide and limulus-positive glycolipid.

Table 22
Production of TNF by macrophages due to the action of the extract of fermented rice powder and inhibitory effect of polymyxin b (TNF-inducing activity of the extract of fermented rice powder)
The concentration of the sample
(ng/ml)
Extract of fermented rice powder
with the addition of polymyxin b
Extract of fermented rice powder
without the addition of polymyxin b
limulus-positive glycolipid with the addition of polymyxin blimulus-positive glycolipid without the addition of polymyxin b
00000
1 0000,1
100002,2
10000,106,1
10000,10,6023,9
100000,52,4029,3

N. Examples of the extract of FERMENTED BROWN SEAWEED

Example 22

Obtaining an extract of fermented brown seaweed mekabu

(1) 1.0 l of water, 0.2 g of potassium dihydrophosphate and 1.15 g of sodium hydrogen phosphate, 8 g of common salt and 0.2 g of potassium chloride were added to a 2-liter conical flask.

(2) Dried brown seaweed mekabu (20 g) was added to (1).

(3) (2) sterilized by autoclaving.

(4) Preparation of inoculum: a single colony ofPantoea agglomeransisolated from wheat flour was added to 5 ml of 2%medium brown seaweed mekabu, pre-prepared with the same composition, and fermentatively PR is 37°C overnight (15 hours) with careful stirring to prepare the inoculum for the fermentation of brown seaweed mekabu.

(5) the Total number (4) was added to (3) and fermentatively at 37°C for 72 hours accurate mixing.

(6) a Solution of fermented brown seaweed mekabu (5) was extracted by heating at 120°C for 20 minutes in an autoclave. Centrifuged (Kubota 8800, 2000 rpm, 10 minutes) and supernatant was collected to obtain an extract of fermented brown seaweed mekabu.

(7) Determination of the content of limulus-active substances limulus-analysis: to determine the system was used Toh-color provided by Seikagaku Corporation, and Seikagaku Corporation Et-1 was used as the standard limulus-active substances. The content of limulus-active substances in the extract of fermented brown seaweed mekabu amounted to 132 ág/ml.

Example 23

Immunostimulatory effect of the extract of fermented brown seaweed mekabu

Cell line acute myelogenous leukemia TNR-1 (1×106/250 μl of RPMI1640 medium containing 10% fetal calf serum), used as human macrophages were placed in a 48-well plate and pre-cultured for 30 minutes. Next, 250 μl of medium (final volume 500 μl) was added so that the final concentration of each sample was 1-10000 ng/ml, the Samples were supplied by the group containing polymyxin B (12.5 µg/ml). After culturing for 4 hours collecting supernatant cultures and cells. The activity of TNF in supernatants was measured using a cytotoxicity test using L-929. The results are presented in table 23. Macrophages were produced TNF even in the presence of polymyxin b under the influence of the extract of fermented brown seaweed mekabu, but in the presence of polymyxin b In macrophages could not produce TNF under the influence of low molecular weight lipopolysaccharide. From this it is evident that the extract of fermented brown seaweed mekabu has biological activity that is different from the one that has a low molecular weight lipopolysaccharide and limulus-positive glycolipid.

Table 23
Production of TNF by macrophages due to the action of the extract of fermented brown seaweed mekabu and inhibitory effect of polymyxin b (TNF-inducing activity of the extract of fermented brown seaweed mekabu)
Concentration of sample (ng/ml)Extract of fermented brown seaweed mekabu with the addition of polymyxin bExtract of fermented brown seaweed mekabu without the addition of polymyxin blimulus-positive glycolipid with the addition of polymyxin bliulus-positive glycolipid without the addition of polymyxin b
00000
10000,1
100002,2
10003,206,1
10002,414.4V013,9
1000018,7of 31.8029,3

Industrial applicability

In accordance with the present invention it becomes possible to inexpensively produce a fermented extract of a plant that is safe immunostimulant. Extract of fermented plants, thus obtained, can be used in medicinal preparations, drugs for animals, quasilogarithmic, cosmetics, products, PI is Denmark, functional food products, feed products and detergents for mammals, including humans (especially domestic animals, Pets and the like), birds (especially grown Chicks, poultry and the like), amphibians, reptiles, fish (especially farmed fish, pet fish, etc. and invertebrates.

1. The method of fermentation of plant material and cultivation of bacteria to extract fermented plant material, comprising preparing a nutrient medium containing plant material, the fermentation of plant material with facultative anaerobic gram-negative bacterium and the simultaneous cultivation of bacteria in the medium with the subsequent receipt of the extract with water or saline buffer and, optionally, drying the obtained extract, and plant material derived from edible plant material containing carbohydrates of which the main component is a polysaccharide, and this bacterium exists in symbiosis with only edible plant material.

2. The method according to claim 1, where the material is entirely edible.

3. The method according to claim 1, where the carbon source, the use of starch.

4. The method according to claim 1, where the specified facultative anaerobic gram-negative bacterium is a Bacillus.

5. methods according to claim 4, where specified facultative anaerobic Bacillus belongs to the family Enterobacteriaceae.

6. The method according to claim 4, where the specified facultative anaerobic Bacillus belongs to the genus Pantoea, Serratia or Enterobacter.

7. The method according to claim 4, where the specified facultative anaerobic Bacillus is Pantoea agglomerans.

8. The method according to any one of claims 1 to 7, where specified edible plant material selected from the grain product, seaweed or beans.

9. The method of claim 8, where the specified edible plant material is a grain product and the specified material obtained from the grain product, choose from wheat flour, rice powder, powder wheat bran, rice bran and sediment from sake.

10. The method of claim 8, where the specified edible plant material is seaweed and specified material derived from seaweed, choose from a powder brown seaweed, powder "mekabu and kelp powder.

11. The method of claim 8, where the specified edible plant material are beans and material derived from soy beans, are a waste fermenting the beans.

12. Extract of fermented plant material obtained by the method according to claim 1.

13. Extract of fermented plant material indicated in paragraph 12 shows the physico-chemical properties, which include the ability to activate macrophages even in p is outstay polymyxin b C.

14. Extract of fermented plant material according to item 12 or 13, which has immunostimulirutuyu activity.

15. The powder extract of fermented plant material obtained from an extract of fermented plant material according to item 12.

16. Application of the extract of fermented plant material by 12 to get drugs, drugs for animals, quasilocal, cosmetics, food, functional food products, feed products and detergents.

17. The use of a powder extract of fermented plant material by 15 to obtain drugs, drugs for animals, quasilocal, cosmetics, food, functional food products, feed products and detergents.



 

Same patents:

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SUBSTANCE: improved methods of obtaining, separating and purifying epothilone B involve fermentation of microbial strain Sorangium cellulosum and, in particular strain ATCC No. PTA 3880 or ATCC No. PTA 3881, collection of resin with adsorbed epothilone B, solvent extraction for separating epothilone B and crystallisation of the desired product. The method involves obtaining epothilone B derivatives. The invention also relates to strains which produce epothilone B and to a method of culturing these strains to produce epothilone B. Methods are disclosed for purifying epothilone B using high-resolution liquid chromatography with reversed or normal phase.

EFFECT: increased ratio of epothilone B to epothilone A due to introduction of admixtures for fermentation, chosen from propionate, propionic acid or some other propionate precursor, and said purification methods allow for production of virtually pure epothilone B.

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FIELD: medicine.

SUBSTANCE: invention can be used for extracting bioluminescent bacteria from sea water. Growth medium consists of sea water taken in the extraction area of bioluminescent bacteria and dry nutrient agar. Medium pH is 7.2-7.4.

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FIELD: chemistry.

SUBSTANCE: colonies of the strain Lactobacillus fermentum 90 TS-4 (21) (All-Russian collection of industrial microorganisms B-7573) selected on grounds of agglutination with 0.1% concanavaline solution in concentration not less than 1.75·10-3 on liquid media MPC-1 are incubated. The cells are separated by centrifugation and obtained cultural liquor supernatant is concentrated over the membrane UM-20 "Diaflo". Obtained supernatant is removed and target product is purified and separated by method of affinity chromatography on concanavaline A-sepharose.

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6 cl, 1 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: preparate pepresents the mixture of lactic acid bacteria strains: Lactobacillus sakei All-Russian collection of industrial microorganisms B-8936 (LSK-104), Lactobacillus plantarum All-Russian collection of industrial microorganisms B-1616 (22/2), Staphylococcus xylosus All-Russian collection of industrial microorganisms B-8945 (SPHYX-45), Pediococcus pentosaceus All-Russian collection of industrial microorganisms B- 8955 (PDA-55) separated from uncooked smoked sausages taken in equal percentage.

EFFECT: transformation of meat raw material to compounds determining its properties, colour formation and enhancing of its nutrition and biological value, decrease of biogenic amines content in ready product.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention can be used in bacteriological tests for identification of bacteria Pseudomonas and manufacturing of nutrient mediums for these tests. The method involves preparation of the nutrient medium by dissolution of L-glutamine or L-glutamic acid (L-glutamate sodium), NaCl, Na2SO4, KH2PO4, K2HPO4, MgSO4·7H2O, agar in specified amount when boiled in 1 litre of distilled water. Glycerol in specified amount is added. The prepared nutrient medium is boiled within 5 minutes. Herewith, medium pH is 7.0±0.2. It is followed with inoculation of day culture Pseudomonas in said nutrient medium. The inoculations are incubated within 24-48 hours at temperature 28°C, then irradiated with UV light and analysed for fluorescein considering yellow-green fluorescence of bacterial lawn and surrounding zone of the nutrient medium.

EFFECT: simplified method for preparing the nutrient medium herewith preserving its nutritious properties and increasing commonality characteristics.

4 ex

FIELD: medicine.

SUBSTANCE: nutrient medium contains pancreatic digest of fresh-water fish, pancreatic digest of infusion broth waste, agar, sodium chloride, sodium carbonate, lithium chloride, vitamin B1, esculin, ammoniacal ferrum citrate, polymyxin B sulphate, ceftazidime, acriflavine hydrochloride and distilled water.

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3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: nutrient medium contains pumpkin water, ammonium phosphate twice-substituted by (NH4)2HPO4, sodium chloride (NaCl), microbiological agar and boiled tap water.

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1 tbl, 4 ex

FIELD: medicine.

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1 tbl, 4 ex

FIELD: medicine.

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3 cl, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: BCG strain is cultivated with the prepared culture sonicated. Further antigenic complex representing cytoplasmic and cell membranes is recovered from said sonicated culture. At 15000 rpm, made supernatant fluid (namely the antigenic complex) is mixed with formalin. The reaction mixture is incubated at temperature 37°C in a thermostat, analysed for protein content, and conjugated with polyvinylpyrrolidone (PVP) in mass ratio of protein 1 mg/ml - PVP 600 mg (1:600) in a magnetic stirrer at a room temperature until homogeneous liquid is made.

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6 tbl, 1 ex

Vaginal composition // 2369389

FIELD: medicine.

SUBSTANCE: vaginal composition comprises viable microorganisms of lactobacillus and/or bifid bacteria, preferentially Lactobacillus bifidus, devitalised saccharomyces, preferentially Saccharomyces cerevisiae, saccharide(s), vitamin A retinal and zinc with specified ratio of composition components.

EFFECT: vaginal protection without negative effect or infliction of harm.

14 cl, 2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention is a skin moistening agent for oral delivery comprising fermented milk whey as active component and obtained by milk fermentation using the strain Lactobacillus, helveticus, CM-4 (deposited with International depositary institution for patents by National Institute of advanced science and technology under inventory number FERM BP-6060).

EFFECT: invention provides skin moistening.

6 cl, 2 ex, 5 tbl

FIELD: medicine.

SUBSTANCE: pharmacological preparations are applied for treatment of purulent wounds and burns, pyoinflammatory dermatopathies, and for acceleration of healing and improvement of wound healing conditions. The external and local pharmaceutical composition possesses antibacterial and necrolytic action; it contains an active complex of bacteriolytic and proteolytic enzymes - Lisoamidase, a base and at least one desired additive chosen from: a substance disarranging the membrane structure of gram-negative microorganisms, an antibiotic, an anesthetic, a reparative process stimulator. The base is hydrophilic substances and a perfluororganic compound emulsion or their physiologically acceptable mixture or their physiologically comprehensible mix in certain component ratio.

EFFECT: enhanced therapeutic activity and ease of use, lower consumption of the active substance, ensured anaesthetising effect, additional stimulation of reparative processes, improved microcirculation, reduced secondary tissue necrosis surrounding a wound caused by excess free radical oxidation.

7 cl, 12 ex

FIELD: medicine.

SUBSTANCE: antibacterial and necrolytic pharmacological composition contains an active complex of bacteriolytic and proteolytic enzymes - Lisoamidase and a base. The pharmacological composition is characterised that as a base, it contains hydrophilic substances or physiologically acceptable mixture thereof providing soft formulation of the composition. Its components are taken in certain mass ratio.

EFFECT: extended application, enhanced therapeutic activity and ease of use, lower consumption of the active substance, ensured anaesthetising effect, improved microcirculation in wound tissues, control of inflammatory processes, and additional stimulation of reparative processes.

6 cl, 14 ex

FIELD: medicine.

SUBSTANCE: external and local pharmaceutical composition possesses antibacterial and necrolytic action, contains an active complex of bacteriolytic and proteolytic enzymes - Lisoamidase, a base and at least one desired additive chosen from: a substance disarranging the membrane structure of gram-negative microorganisms, an antibiotic, an anesthetic, a reparative process stimulator or physiologically acceptable mixture thereof. The components are taken in certain mass ratio.

EFFECT: extended application, enhanced therapeutic activity and ease of use, lower consumption of the active substance, ensured anaesthetising effect, additional stimulation of reparative processes, improved microcirculation, reduced secondary tissue necrosis surrounding a wound caused by excess free radical oxidation.

7 cl, 14 ex

FIELD: medicine.

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EFFECT: higher vaginal cleanness and recovery of normal flora, discomfort elimination, improvement of vaginal hygienics ensured by inhibition of pathogenic and opportunistic vaginal flora providing antiinflammatory and immunocorrelating action.

4 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: treating allergies is ensured by introduction of antiprotozoan preparations and probiotics, including at first "Balance Narine-F", then "Narine". To prevent re-infection of the patient, antiprotozoan preparations are used to treat persons being in contact with him.

EFFECT: reduced time and improved clinical effectiveness due to normalisation of microbial balance and recovery of enzyme intestinal system that acts the important part in allergic pathogenesis.

1 tbl, 2 ex

FIELD: food industry.

SUBSTANCE: invention is referred to feeding stuff production. Fodder dietary supplement for piglets contains fat-free milk, biphidus bacteria B. Bifidum, as bacterial compound "Bifidumbacterin" additionally contains dry leafy mass wt %: Fat-free milk 80; bacterial compound "Bifidumbacterin" 10, dry leafy mass 10.

EFFECT: fodder supplement optimises feeding rations and thus helps to normalise microbiocenosis connected to bifidogenic activity disorder, increase fermentative activity and fodder digestibility, improve carbohydrate metabolism, increase productivity, provide pigs and environment ecological health, increase fodder digestibility, improve livestock stability.

4 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: to activate growth of bifidus bacteria, concentrated kvass wort in amount 0.1-10 wt % is introduced into an agitated nutrient medium before sterilisation thereof. Bifidus bacteria are cultivated at temperature 37-38°C during 6-12 h to prepare a bacterial mass of bacteria content 108 CFU/ml and more. There are applied consortium of strains Bifidobacterium bifidum 1, Bifidobacterium bifidum All-Russian collection of industrial microorganisms B-3300, Bifidobacterium bifidum All-Russian collection of industrial microorganisms B-3299, Bifidobacterium longum All-Russian collection of industrial microorganisms B-2000, Bifidobacterium longum All-Russian collection of industrial microorganisms B-3301, Bifidobacterium longum All-Russian collection of industrial microorganisms B-5894 or consortium of same strains additionally containing Bifidobacterium adolescentis All-Russian collection of industrial microorganisms B-5894.

EFFECT: possibility to make cultured milk foods, fermented and unfermented, hygienic and cosmetic means, biologically active additives and bacterial preparations containing Bifidus bacteria in shorter terms with high concentration of industrial biomass.

3 cl, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely paediatrics, pulmonology, and covers treatment of recurrent stenosing laryngotracheistis in children. It is enabled by determination of extent of disbiotic conditions of nasopharynx and oropharynx membrane. The first extent requires introduction of Bronchomunal, Bifiform, Fenspirid and retinol acetate, while in the second extent, Ribomunyl, Bifiform, Fenspirid, Triovit and Phenibut are prescribed.

EFFECT: method provides selection of optimum treatment regimen considering extent of nasopharynx and oropharynx membrane disbiosis, normalised indices of breathing function and airway sensitivity.

2 dwg, 5 tbl, 3 ex

FIELD: food industry.

SUBSTANCE: invention relates to soluble food fibers (made of oat or barley grain) extraction method and treatment by fermentative hydrolysis. Milled oat or barley grain and any of its fractions combined by endosperm which are enriched by β-glucans are combined and dispersed in water without additional thermal treatment. After that it is fermentative process using enzymes which decompose starch with following possible stage of deactivation of enzymes by wet thermal treatment. After that hydrolysate mixture inadvertently or by centrifugal processing is divided into at least 3 separate fractions. The first fraction contains soluble food fibers complex, contains more than 20% of β-glucans as dry substance, second water fraction and third fraction containing most part of protein and oil together with dissoluble fiber material of milled grain. Besides the real invention relates to usage of obtained β-glucans.

EFFECT: proposed method allows to separate clean fraction, enriched with β-glucans.

28 cl, 22 ex

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