Controlled activation of reuterin induction

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and is used to preparing the cell cultures of Lactobacillus reuteri, containing reuterin to be kept inside the cells. The method involves the fermentation of cell cultures, the addition of 1,2-propanediol or glycerol to the reuterin-producing cell systems Lactobacillus reuteri in the beginning of the fermentation, the addition of glycerol to the cell cultures of Lactobacillus reuteri during the production and preservation of the cells Lactobacillus reuteri. The product prepared by the declared method contains the kept cells Lactobacillus reuteri with reuterin kept in the cells.

EFFECT: group of inventions allows producing the large amounts of reutrin-loaded Lactobacillus reuteri to be used for the purpose of both preventing and treating diseases, and as a food additive.

9 cl, 9 dwg, 5 ex

 

The technical FIELD

The invention relates to a controlled activation system production reuterin Lactobacillus reuteri by adding glycerin and other substances in the receiving cell cultures, and save the resulting reuterin in the bacterial cell during accumulation and storage. In particular, the present invention relates to the production of large quantities of L.reuteri, loaded reuterin, and the use of such loaded bacteria for such purposes as the prevention and treatment of diseases, as a food additive, etc.

The LEVEL of TECHNOLOGY

Usually the cells of prokaryotes belong to the primitive, although some of them contain an unusual shell, known as microcompartment (MCS), which, as it turned out, represent primitive organelles inside bacterial cells. Carboxysome (which is involved in the fixation of carbon dioxide) for nearly 30 years was the only recognized microcompartments in microbial cells.

In 2005 Professor Todd O. Yeates and his colleagues discovered the first structural properties of microcompartments bacteria. Upon receipt of the first structures at high resolution bacterial proteins microcompartments were identified rules of the Assembly, very similar to those observed in some viruses. Six identical protein subgroups are combined into a review of the unit, which was before the hat is basic element in the formation of the shell. These review units closely Packed with each other, forming a molecular layer containing only tiny pores. This dense packing, apparently, limits the movement of molecules into and out of microcompartments, except for long.

Cluster analysis of homologous substances of microbial microcompartments - specific proteins, showed that these membranes are involved in at least seven different metabolic processes in bacteria of different species (Thomas A. Bobik. 2007. Bacterial Microcompartments. Microbe. 1:25-31). Elementary links of bacterial microcompartments consist of proteins and glycoproteins. Electron microscopy (used for research microcompartments) shows the absence of lipid mono - or bi-layer (as in the membranes of eukaryotes)surrounding such microcompartment, making them the only known protein metabolic organelles in living cells. A number of bacterial genera, including Salmonella, Escherichia, Klebsiella, Clostridium, Fusobacterium, Shigella, Listeria and Yersinia, contain the components necessary for the decomposition of 1,2-propane diol (1,2-PD) or ethanolamine, microcompartment (1). Another property of microcompartments, as expected, is their ability to act as a reservoir for toxic to bacteria substrates, as in the case of c antimicrobial reuterin at L.reuteri.

the search in GenBank, conducted Bobik, genes shell microcompartments showed that approximately 25% (85 of 337) bacterial genomes contain homologous genes shell. Most of these 25% of bacterial genomes that carry such homologous genes, the genes of the shell form a cluster with other genes encoding enzymes related to microcompartment. It showed that the genes encoding the production of reuterin, and genes encoding structure microcompartments are adjacent.

In may 2008 Sriramulu et al. (Sriramulu DD, Liang M, Hernandez-Romero D, Raux - Deery E, Lunsdorf H, Parsons JB, Warren MJ, Prentice MB: Lactobacillus reuteri DSM 20016 produces cobalamin-dependent diol dehydratase in metabolosomes and metabolizes 1,2-propanediol by disproportionation. J Bacteriol 2008, 190(13):4559-4567) presented the first evidence that producing antibacterial agent organism Lactobacillus reuteri has possibilities for synthesis of bacterial microcompartment (carboxysome or metabolome) when grown in modified MRS medium containing 65 mm 1,2-propane diol (PD) and a small amount of glucose. The body was produced kobulnicky the enzyme dialdehydes induced 1,2-PD. Operons paired synthesis of cobalamin and pdu (recycling propane diol) was present in the genomic sequence DSM 20016 L.reuteri, and full pdu-operon from the laboratory strain DSM 20016 L.reuteri was amplified PCR, confirming its presence in propane the IOL-metabolizing organism. However, growing in modified MRS medium with 65 mm 1,2-PD and a small amount of glucose can not be used on an industrial scale because of the very low growth rate of bacteria in this environment. Unlike the present invention, there is not disclosed the addition of glycerol during the production of cell cultures, which provides load microcompartments reuterin.

Lactobacillus reuteri is a well-known bacterium, producing an antimicrobial substance 3-hydroxypropylmethyl (HPA), also called deuterium. Antibacterial activity reuterin described, for example, in U.S. patent No 5439678, 5458875, 5534253, 5837238 and 5849289. Reuterin is a low molecular weight, neutral, water-soluble compound capable of inhibiting the growth of bacteria of all genera and species studied to date, including: Escherichia, Salmonella, Shigella, Proteus, Pseudomonas, Clostridium, Staphylococcus, Streptococcus and Helicobacter pylori, as well as some fungi and other microorganisms. By using glycerol as an external electron acceptor, it is converted into 1,3-propandiol, this produces reuterin as intermediate. This reaction takes place in conjunction with the fermentation of sugars such as glucose.

Getting reuterin depend on a complex system, comprising: an enzyme glycerol/diol-dehydratase catalyzing the reaction; cobalamin (vitamin B12), copact the R enzyme, which is synthesized through the complex; factors used for the regeneration of the enzyme; and structures microcompartments with the transverse size of more than 100 nm and formed by polypeptides. All of the above encoded in more than 50 genes that are induced in case they are needed. The addition of 1,2-PD or glycerol in the culture medium leads to the creation of a large number of structures microcompartments. The addition of glycerol at a later stage in the bacterial culture results reuterin. Reuterin is produced, collected and stored inside microcompartments, as long as they will not release the substance. In the absence of growth and substrate (including glycerol) when applying L.reuteri in normal condition (without a fully loaded microcompartments), for example, on the skin, the release reuterin does not usually occur. Thus, what was unexpected was activated L.reuteri with reuterin accumulated in microcompartment, in accordance with the invention successfully and quickly released reuterin in adverse environments, such as human skin, food, or other such places, and, of course, in more traditional places for probiotics, such as the digestive tract and the IMP, mouth and nose of animals, including humans.

The growth of skin pathogens such as Staphylococcus aureus, Streptococcuspyogenes or Propionibacterium acnes, or some yeast can lead to infection of wounds and disturbance of the skin system, or even to more serious disorders of the skin or mucous membranes, such as eczema, candidiasis, dermatitis, impetigo, etc. For the treatment of these pathogenic agents known to many tools. Most are traditional antibiotics or antibacterial chemical components. As such, for example, you can specify the composition based on aldehydes and their derivatives.

Another skin disease, where treatment includes both oral and local antibiotics, is a rosacea damaging the middle part of the face, causing persistent redness of the face and nose.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and related gram-positive pathogens are a growing problem in medicine. These include MRSA, methicillin-resistant Staphylococcus epidermidis (MRSE), and methicillin-resistant coagulated negative Susceptible (MRCNS). Vancomycin, glycopeptides antibiotic, is currently the drug of choice to combat these infections. With the increased use of vancomycin should expect the emergence of vancomycin-resistant strains are Susceptible (to the VRSA). Thus, there is a growing need for tools that are effective against such pathogens (MRSA/to the VRSA), but it does not provide regulate is inoe side effects.

S.aureus usually colonizers nostrils, although the respiratory tract, open wounds, intravenous catheters and urinary tract are also potential sites for infection. Healthy individuals can be asymptomatic carriers of MRSA during the period from several weeks to many years.

Another example of skin disorders that are difficult to treat and which may be caused by a wide number of reasons, is contact dermatitis, which in sensitive subjects may begin when skin contact with the external stimulus/agent.

The use of Lactobacillus for the treatment of skin disorders is already known in this field, for example, disclosed in patent application U.S. No 05/201996. The invention relates to the prevention and/or treatment of skin disorders with the use of preparations containing probiotic bacteria such as Lactobacillus fermentum strain VRI-002. The preferred route of administration is oral.

Other bacterial agents, such as Bacillus, can also be used for skin or mucous membranes. More specifically, in the application WO 98/47374 strain of Bacillus used in compositions intended for the prevention of bacterial, viral or fungal skin infections.

However, there is a problem arising from the application of lactic acid bacteria in the local application or other adverse the environments with other bacteria, described in the prior art, which is the short life of bacteria caused by adverse environment on the skin or elsewhere. This problem is solved by the present invention, through the introduction of L.reuteri with the loaded microcompartments, in the “stand by” (waiting) for secretion reuterin. Thus, L.reuteri performs secretion reuterin until death.

BRIEF description of the INVENTION

This invention relates to a method of controlled activation system production reuterin L.reuteri by filling the system producing reuterin 1,2 PD or glycerine and then, upon receipt of cell cultures, addition of glycerol to the culture of bacteria at some point before accumulation. This invention also relates to the addition of vitamin B12, cobalt and vitamin C in the culture medium to improve conditions for optimal growth and formation of microcompartments and reuterin bacteria L.reuteri during retrieval.

In particular, this invention relates to the production of a large number of cells L.reuteri, which are loaded with deuterium, and the use of such bacteria obtained in compositions, for example, for the prevention and treatment of diseases and in food compositions. More specifically, such compositions are intended for administration to humans, for example, topically, for prophylact the key or disorders, caused by pathogens of the skin system. These compositions can also be used for nasal use for the treatment of MRSA. The invention overcomes the problem of growth and survival of bacteria L.reuteri in adverse environments.

Accordingly, before disclosing certain embodiment of the invention, it should be considered that the invention is not limited in this application to the details of construction and sequence of components specified in the following description or shown in the figures. The invention covers other ways to perform and can be carried out and performed in a variety of ways. In addition, it should be understood that the phraseology and terminology used here for descriptive purposes and should not be construed as limiting.

BRIEF DESCRIPTION of FIGURES

Figure 1. Shown are histograms illustrating the concentration reuterin in the supernatant obtained from strains DSM 17938 (empty rectangles) and MM4-1 (black squares)grown to stationary phase in B12 environment in the presence or absence of vitamin B12 (top) or cobalt (below).

Figure 2. Shows a histogram illustrating the effects at concentrations of supernatants obtained from strains DSM 17938 and MM4-1, grown to stationary phase in MRS medium.

Figa. Shows a histogram illustrating the effect of adding 1,2-PD on to the concentrations reuterin in the supernatant and the survival of strain MM4-1, grown to stationary phase in MRS without added cobalt (A, D), vitamin B12 (B, E), with the addition of 50 ng/ml of cobalt (C, F), 1 μg/ml of vitamin B12. Black rectangle shows the absence of added 1,2-PD. The white rectangle shows the addition of 65 mm 1,2-PD. Gray rectangles shows the addition of 200 mm of 1,2-PD. Survival was measured before (0 h) and after (2 h) incubation of the cells in 200 mm glycerol/water solution.

Fig.3b. Shows a histogram illustrating the effect of adding 1,2-PD on the concentration reuterin in the supernatant and the survival of strain DSM 17938, grown to stationary phase in B12 environment with the addition of 1 μg/ml of vitamin B12. Black rectangle shows the absence of added 1,2-PD. The white rectangle shows the addition of 65 mm 1,2-PD. Gray rectangles shows the addition of 200 mm of 1,2-PD. Survival was measured before (0 h) and after (2 h) incubation of the cells in 200 mm glycerol/water solution.

Figure 4. The picture shown MCS obtained by transmission electron microscopy (TEM). Strains DSM 17938 (A) and MM4-1A (B) were grown in MRS. Strains DSM 17938 (A) and MM4-1A (B) were grown in MRS with added vitamin B12 (1 mg/ml) and 200 mm 1,2-PD. White arrows indicate MCS formed in bacteria.

Figa. Shows concentration reuterin in the supernatant obtained from cells MM4-1A, after exposure to 200 mm glycerol aqueous solution within 45 minutes Bacteria were grown in B12-medium (50 ng/ml cobalt) with the addition of 200 mm 1,2-PD, 200 mm glycerol or without the addition of these components.

Fig.5b. Shows concentration reuterin (chart above) in the supernatant obtained from cells MM4-1, after exposure to 200 mm glycerol aqueous solution for 45 minutes Histogram below shows the concentration reuterin in cellular precipitate after exposure to 200 mm glycerol aqueous solution for 45 min, the Bacteria were grown in MRS (1), in MRS with the addition of 1 μg/ml vitamin B12 (2), in MRS with the addition of 1 μg/ml of vitamin B12 and 200 mm 1,2-PD (3), in MRS with the addition of 1 μg/ml of vitamin B12 and 200 mm glycerol (4) and in MRS with the addition of 1 μg/ml of vitamin B12 and 500 mm glycerol.

6. Shows a snapshot of the MAS-NMR spectrum (top) of the detected substances associated with cells MM4-1, after washing the cells and exposure to 200 mm glycerol aqueous solution for 45 minutes. Cells were grown in MRS with the addition of 1 μg/ml of vitamin B12 and 200 mm glycerol. The arrow indicates the aldehyde group in the 3-HPA. The image below shows the substances associated with obtaining reuterin. Substances marked with an asterisk were found MAS-NMR.

7. The histogram shows that the presence of sucrose does not affect the production reuterin.

DETAILED description of the INVENTION

Obtaining cultures L.reuteri, which will be used as the E. probiotics spend without glycerol and then lyophilizer. In these bacteria the system used to obtain reuterin is not activated, but in favorable conditions, the system can be activated through 30-60 minutes after bacteria will come in contact with glycerol. In adverse conditions such activation may take much longer or not happen at all.

In cases where you want a product containing L.reuteri with fast production reuterin, or where conditions for growth L.reuteri adverse, culture L.reuteri can be improved by the addition of glycerol during culture. Glycerin (1-500 mm) can be added during the stage of fermentation, or it may be added together with cryoprotectants after fermenting and washing, if it is required, but before lyophilization. The receiving system reuterin, including the formation of microcompartments L.reuteri, can be improved by adding to the system of production reuterin 1,2 PD or glycerin in the beginning of fermentation.

The product of the cell culture can be obtained in several ways, including without limitation the three different options below.

1. Freeze-dried product containing cells L.reuteri, converts glycerol to reuterin at the end of the fermentation stage of the production method, but at the stage of lyophilization. PR is the product, thus obtained will contain lyophilized cells and reuterin, both inside and around cells. In this embodiment of the method will be derived bacteria Laden with deuterium.

2. The second option is similar to 1, but to the system of production reuterin bacteria add 1,2 PD or glycerol, and, possibly, cobalt or vitamin B12 in the early stages of fermentation. In this embodiment of the method will be obtained lyophilized bacteria Laden with deuterium and able to produce and accumulate reuterin.

3. Liofilizirovanny product containing cells L.reuteri, converts glycerol to reuterin after fermenting and washing, if needed, adding glycerin and producing reuterin for about 30-45 minutes at 37°C before being freeze-drying. Glycerol for producing reuterin can, for example, to add together with cryoprotectants. System production reuterin bacteria filled 1,2 PD or glycerine in the early stages of fermentation. Advantages of option 3 ways to obtain compared to 2 is that option 3 is more convenient for a number of industrial installations and is more suitable for control education reuterin.

The addition of 1,2-PD or glycerol in the culture medium affects both the survival and which is of MCS. Enzyme complex PduCDE responsible for the conversion of 1,2-PD in Propionaldehyde, is also responsible for the conversion of glycerol in reuterin, which opens up the opportunity that the MCS, which are formed when growing bacteria in the presence of 1,2-PD, can also work as a "factory" to obtain reuterin, when the contact of bacteria with glycerin and lack of funds for subsequent metabolism reuterin (i.e. bacteria are in a stationary phase or exposed to glycerol in water solution). Reuterin, which is formed in MCS, accumulates in the cells in larger quantities compared to the cell without MCS. This allows you to get "loaded" reuterin bacteria, for example, prior to lyophilization.

Observations Sriramulu et al. for strain DSM 20016 authors repeated for strains MM4-IA and DSM 17938. However, growing in modified MRS medium with 65 mm 1,2-PD and a low amount of glucose is not suitable for industrial scale because of the very low growth rate of bacteria in this environment. Instead, the authors have added 200 mm 1,2-PD and 1 μg/ml of vitamin B12 in unmodified MRS environment and studied bacteria for producing visible MCS after cultivation for 24 h at 37°C (used electron microscopy to visualize). As MM4-1A and DSM 17938 strains were produced MCS in these conditions (Figure 4).

Glycerin, similar to 1,2-PD, the cells metabolism and regrowth is regulated by the same enzyme complex, called PduCDE thus, it is also possible to use glycerol to induce the formation of MCS in bacteria, similar to that observed for 1,2-PD. Growth of strain MM4-1A or 200 mm glycerol or 1,2-PD results in cells that behave identically in the formation reuterin and its relationship to sediment bacterial cells after exposure for 1 h solution of glycerol on bacteria (Figa and 5b).

In addition to the flushing of sediment (see above, Fig.5b) the authors also investigated the washed cell precipitate on the content reuterin using MAS-NMR. Then strain MM4-1 were grown to stationary phase in MRS medium with the addition of B12 (1 mg/l) and 200 mm 1,2-PD. Then the grown bacteria were subjected to 200 mm glycerol in aqueous solution, and incubated for 1 hour at 37°C. the Cells were left on ice and washed 2 times deuterium water (D2O)containing 200 mm glycerol. The residue (approximately 20 μl of the wet weight)obtained at final stage, was dissolved in 20 μl of D2O without glycerol and measured the content reuterin method MAS-NMR. Using this technique, the authors found two of the three species reuterin and some decomposition products reuterin and 1,2-PD (Figa and 6b).

It is shown that the addition of glycerol and 1,2-PD add some other substances in the culture medium can affect the survival of CL is current, the formation of MCS, producing reuterin and adaptability of bacteria, such substances are, for example, vitamin B12, cobalt and vitamin C.

To show that the addition of vitamin B12 or cobalt in the culture medium affects the adaptability of L.reuteri, investigated two different types of media for growing, MRS (Oxoid) and B12 environment for research (Fluka)in respect of production reuterin and adaptability strains L.reuteri DSM 17938 and MM4-1 A. the Main difference between the two environments for growing is that MRS contains an undefined vitamins added in the form of yeast extract, whereas B12 environment for research (hereinafter referred to as B12-environment) contains a specific composition of all vitamins necessary for the functioning of bacteria, except for B12, which is missing. Another important difference in the composition of vitamins between these two environments is that B12-environment includes vitamin C in the amount of 4 g/l, possibly several times more, than MRS.

The authors used B12 environment as a tool for monitoring reuterin, forming microcompartments (MCS) and the adaptability of bacteria adding vitamin B12 or cobalt. Vitamin B12 is an essential component of the enzyme complex PduCDE, which is responsible for the conversion of glycerol in reuterin. For real the sale of its biological functions of vitamin B12 requires the presence of ion cobalt. If you have a cobalt, but not vitamin B12, reuterin can be formed only if the downregulation of genes cob-operon, as they are essential for the formation of cobalt containing molecules B12. Because the expression of cob-operon is associated with the expression previously located pdu-operon, perhaps through regulator PocR (Santos F, Vera JL, van der Heijden R, Valdez G, de Vos WM, Sesma F, Hugenholtz J: The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL 1098. Microbiology 2008, 154(Pt 1):81-93; Cheng S, Liu Y, Crowley CS, Yeates TO, Bobik TA: Bacterial microcompartments: their properties and paradoxes. Bioessays 2008, 30(11-12): 1084-1095), the authors investigated the production reuterin and adaptability adding vitamin B12 or cobalt in MRS or B12 environment.

Shown a significant impact on both strain (DSM 17938 and MM4-1A) in relation to the production reuterin when grown in B12 environment by changing the number and / or vitamin B12 or cobalt (Figure 1). This is logical, because the production reuterin cannot occur without exposure of B12 molecules that are or should be added directly, or be synthesized by bacteria in the presence of cobalt ions.

Wednesday MRS, unlike B12 environment that already contains some amount of vitamin B12, as added yeast extract contains a mixture of vitamins. The measured production reuterin in MM4-1A and DSM 17938 grown on simple MRS environment, coincided with the eat, which was previously reported in the literature, but MM4-1 A was not reached the same level of production reuterin as strain DSM 17938 (2) (Spinler JK, Taweechotipatr M, Rognerud CL, Ou CN, Tumwasorn S, Versalovic J: Human-derived probiotic Lactobacillus reuteri demonstrate antimicrobial activities targeting diverse enteric bacterial pathogens. Anaerobe 2008, 14(3): 166-171). Unlike B12 environment, adding vitamin B12 or cobalt in MRS medium did not lead to convincing results regarding the increase in production reuterin MM4-1A and DSM 17938.

However, adding vitamin B12 to MRS environment is manifested synergistic effect of 1,2-propane diol (1,2-PD) in respect of the properties adaptability of bacteria MM4-1A (Figure 3). Strengthening adaptability strain MM4-1A grown in MRS supplemented with vitamins B12 and 200 mm 1,2-PD, correlates with a decrease in detected reuterin in the environments surrounding the bacteria (Figure 3). The authors suggest that this effect was due to the fact that the addition of vitamin B12 and 1,2-PD to MRS increased the formation of microcompartments (MCS) in bacteria. The increase generated MCS has led to the fact that reuterin produced accumulates in educated MCS inside bacteria.

For evaluation, the authors showed that adding vitamin B12 and 1,2-PD in normal MRS:

Creates visible MCS in MM4-1A and DSM 17938 (Figure 4). Increase resistance to producing reuterin when growing MM4-1A in MRS with the addition of the of itamin B12 (1 mg/l) and 200 mm 1,2-PD (Figure 3). Increase resistance to producing reuterin when growing DSM 17938 in B12 environment with added vitamin B12 (1 mg/l) and 200 mm 1,2-PD (Fig.3b).

The authors also showed that bacteria MM4-1A grown in this way (or with the addition of glycerin instead of 1,2-PD), accumulate more reuterin inside bacterial cells and less outside (Fig.5b).

Adding vitamin B12 in MRS with additional 1,2-PD only has an effect on strengthening properties MM4-1 to hold the endogenous biogas produced reuterin. The saturation of the environment for the cultivation of vitamin B12 contributes to the formation of a functioning MCS in bacteria when grown in the presence of 1,2-PD (Figure 3 and 3b).

From the obtained L.reuteri, loaded activated microcompartments containing reuterin, can be prepared composition having various forms, usually in the form of creams, lotions, pastes, powders, capsules, tablets, ointments, emulsions, nasal sprays and other Such preparations can be prepared by the known methods using pharmaceutically acceptable carriers, excipients, diluents or aide-de-camps. Such techniques and ingredients known and widely described in the guides and directories.

Bacteria according to the invention can be used to produce compositions intended, for example, for prevention or treatment of disorders associated with pathogens of the skin system, such as Staphylococcus aureus, Streptococcus pyogenes, Propionibacterium acnes, or yeast. Such violations skin may constitute, in particular, acne, atopic dermatitis, candidiasis, impetigo or eczema secondary infection. Disorders of the skin may also have a non-bacterial cause, for example, rosacea, psoriasis, wounds from burn injuries, bedsores and other slow-healing wounds. Bacteria according to the invention can be used to obtain the composition for treating disorders caused by pathogens of the skin system. In particular, the bacteria can be used to obtain a composition for prevention or treatment of MRSA.

The above should be considered only as illustrative of the basic invention. Furthermore, since numerous modifications and changes can easily be performed by a specialist in this area, it is not desired to limit the invention to the specific implementation and the sequence of operation shown and described, and accordingly, all modifications and equivalents falling under the scope of the claims, may be used.

The present invention also provides a pharmaceutical composition comprising the product obtained by the method according to the invention described here.

EXAMPLES

EXAMPLE 1

Obtaining a lyophilized powder L.reuteri loaded with mi is acompartment, containing reuterin activated during stage fermentation

Composition of fermentation medium

Dextrose monohydrate 60 g/l
Yeast extract KAV 20 g/l
Peptone type PS (pork) 20 g/l
The diammonium citrate 5 g/l
Sodium acetate (×3 H2O) 4.7 g/l
Dipotassium hydrogen phosphate, 2 g/l
Tween 80, 0.5 g/l
Silybin (antifoam) 0.14 g/l
Magnesium sulfate 0.10 g/l
Manganese sulfate 0.03 g/l
Zinc sulfate heptahydrate, 0.01 g/l
Water q.s.

Wednesday centrifugation

Peptone 0-24 Orthana (pork)

The cryoprotectants

Lactose (cow) 33%
Hydrolyzed gelatin (bovine) 22%
MSG 22%
Maltodextrin 11%
Ascorbic acid 11%

The stage of obtaining a lyophilized powder of Lactobacillus reuteri.

Twenty ml of medium was inoculable 0.6 ml of lyophilized powder of Lactobacillus reuteri from the working tube of the cell Bank. Fermentation was performed in a flask at 37°C for 18-20 hours without stirring or control pH, i.e. statically.

Two flasks, each containing 1 liter of medium was inoculable 9 ml of cell suspension per liter. Fermentation was performed at 37°C for 20-22 hours without stirring or control pH, i.e. statically.

Two liter of cell suspensions from stage 2 was inoculable 600-liter capacity. Fermentation was performed at 37°C for 13 hours while controlling the pH and stirring. At the beginning of the fermentation the pH was 6.5. The pH control was started, when the pH dropped below 5,4, using 20% sodium hydroxide solution. Brought the pH to 5.5.

Fourth and last is Yuyu stage fermentation was performed in a 15,000-gallon capacity inoculation of the product from step 3. Fermentation was performed at 37°C for 9-12 hours with control of pH and mixing. At the beginning of the fermentation the pH was 6.5. The pH control was started, when the pH dropped below 5,4, using 20% sodium hydroxide solution. Brought the pH to 5.5. 100 mm glycerol was added at the last phase of fermentation, just before the culture reaches stationary phase. Fermentation was completed after reaching the stationary phase of the culture, as shown by a reduction of the added sodium hydroxide solution. Approximately 930 liters of sodium hydroxide solution was added to 10200 litres of environment and 600 liters of inoculum during fermentation.

Cell suspension with the last stage of fermentation was separated twice at 10°C in a continuous centrifuge Alfa Laval. After the first centrifugation, the volume of cell suspension was reduced from approximately 11730 litres up to 1200 litres. This volume was washed 1200 liters of a solution of peptone (Peptone 0-24, Orthana) 3000 litre tank and then separated before being mixed with cryoprotectants. Stage washing with peptone performed to prevent the temperature drops to the freezing point during lyophilization.

After the second centrifugation, the volume of cell suspension was reduced to 495 litres. This volume was mixed with 156 kg of a solution of cryoprotectant, getting about 650 liters of cell suspension.

The cell suspension perekati the Ali in a 1000 liter capacity. Then the tank was moved to the installation for lyophilization.

Installation for lyophilization poured on each plate for lyophilization to 2 litres (exactly) of cell suspension. The maximum capacity of lyophilizate is 600 liters and the remains of the cell suspension were thrown.

Cell suspension Lactobacillus reuteri liofilizirovanny within four to five days and the dry matter content was 18%.

During the process of lyophilization working pressure was between 0,176 mbar and 0.42 mbar. The vacuum pump is started when the pressure reached at 0.42 mbar. PRT (research integrity) was used to determine the end of the process. If the PRT or the increase in pressure was less than 0.02 mbar after 120 s, the process stopped.

EXAMPLE 2

Obtaining a lyophilized powder L.reuteri, with the loaded microcompartments containing reuterin filled and activated during the fermenting

The method of obtaining the same, as described in example 1, but with the addition of an additional 200 mm 1,2-PD, vitamin C (4 g/l) and vitamin B12 (1 mg/ml) in the culture medium.

EXAMPLE 3

Obtaining a lyophilized powder L.reuteri, with the loaded microcompartments containing reuterin filled during the stage of fermentation, and activated for forming reuterin stage before lyophilization

Im the same, as described in example 1, but with the addition of an additional 200 mm 1,2-PD, vitamin C (4 g/l) and vitamin B12 (1 mg/ml) in the culture medium. But 100 mm glycerol added to the cell suspension before transporting to the installation lyophilization instead fermentation phase.

EXAMPLE 4

Obtaining ointment with L.reuteri, with activated system production reuterin

Ointment prepared from the following components:

Dried powder L.reuteri, with activated system production reuterin obtained, for example, any of the industrial methods described above.

Auxiliary substances for the product (anhydrous oil stabilized solid fat or wax). The oil is preferably a vegetable oil such as rapeseed or palm oil.

Solid fat, such as beeswax.

Preservatives and stabilizers, any known in the field of ointments.

The method includes melting solids, and mixing with oil (AkomedPv, [AAK]) and other ingredients. Dried powder L.reuteri was added to the mixture at a temperature below 55°C. the Ointment was obtained by stirring the mixture before hardening.

The tubes were filled with ointment and corked. The prepared ointment contains approximately 10E+08 CFU obtained culture L.reuteri per gram of ointment.

EXAMPLE 5

Treatment of rosacea man

A woman with long life & energy saving is Noah history of rosacea were treated liofilizirovannami cultures L.reuteri, received in accordance the present invention. The subject was treated twice a day, morning and night. Each time the ointment rubbed into the skin, causing a thin layer.

After 2 weeks rosacea has improved markedly without antibiotics that were prescribed to treat the condition. When cancel treatment L.reuteri status was returned, but was suppressed when the continuous application of L.reuteri.

EXAMPLE 6

Obtaining nasal spray

Nasal drug, including L.reuteri, loaded structures microcompartments who are ready for production reuterin can be made in various forms for administration, for example, in the form of a spray, drops, gel, ointment, cream, powder or suspension, using a dispenser or other device, if necessary. Various dispensers and delivery vehicles known in the field, including single dose ampoules, sprays, nebulizers, pumps, nasal patches, nasal sponges, nasal capsules, etc.

In the General case, the drug may be in solid, semi-solid or liquid form. In the case of solid forms components can be connected together in a blender, drum mixer, by lyophilization, evaporation of the solvent, joint grinding, spray drying and other methods known in this field.

Semi-solid preparations suitable for nasal application, which may be in the form of oil gel or ointment.

In a preferred embodiment of nasal drug has a liquid form, which may include oil solution, oil suspension. The liquid preparation may be in the form of a nasal spray or nasal drops, using devices known in the field, including nebulizers, which can deliver the selected volume of drug aerosol form liquid droplets. For example, commercial spray pump with the supplied volume of 50 ál or 100 ál available, for example, from Valois (Congers, N.Y.), with adults and children spray tips.

The liquid preparation may be obtained by known methods. For example, nasal drug can be obtained by mixing L.reuteri, loaded reuterin with oily base, such as pharmaceutically acceptable oil, such as olive oil, lanolin, silicone oil, glycerin fatty acid, etc.

It should be clear that the excipients necessary for obtaining drug stability and/or bioavailability, can be included in the preparation. Typical excipients include sugars (glucose, sorbitol, mannitol, sucrose, penetration enhancers (chitosan), thickeners, and agents that improve stability (cellulose, polyvinylpyrrolidone, starch, etc), buffers, preservatives and/or acids and bases for Regulus in the regulation of pH, etc.

Although the invention has been described with reference to specific examples, a person skilled in the field should be clear that the invention can be implemented in various forms.

1. A method of obtaining cell cultures of Lactobacillus reuteri containing reuterin stored inside the cells, including:
obtaining cell cultures Lactobacillus reuteri, where receiving comprises the fermentation of these cell cultures;
the addition of 1,2-propane diol or glycerol to reuterin-producing systems of cells of Lactobacillus reuteri in the beginning of fermentation;
adding glycerol to the cell cultures Lactobacillus reuteri during retrieval, thereby obtaining cells of Lactobacillus reuteri, where reuterin is stored inside the cells; and maintaining the cells of Lactobacillus reuteri.

2. The method according to claim 1, where the fermentation involves adding glycerol at the end of the fermentation process.

3. The method according to claim 1, where storing cells of Lactobacillus reuteri includes lyophilization cell cultures after fermentation.

4. The method according to claim 2, where the preservation of Lactobacillus reuteri cells includes lyophilization cell cultures after fermentation.

5. The method according to claim 3, further comprising adding at least one cryoprotectant together with glycerol in cell culture after fermenting, before lyophilization.

6. The method according to claim 3, where from about 1 to about 500 mm glycerol added last is the stage of fermentation, but before lyophilization in the production process.

7. The method according to any of claim 2 to 6, where fermentation cell cultures of Lactobacillus reuteri includes adding to the cell cultures at least one of the following: cobalt, vitamin B12, vitamin C, or their combinations in the early stages of fermentation.

8. The product obtained by the method according to any one of claims 1 to 7, where the product contains stored cells of Lactobacillus reuteri, with reuterin is stored in the cells.

9. Product of claim 8 for use in the treatment of disorders caused by pathogens of the skin system.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: method includes cultivation of previously prepared culture of the recombinant strain B. anthracis 55ΔTPA-1Spo-. The cell mass is separated using the filtration module with a membrane having a pore diameter of 0.2 mcm. Protein EA1 is extracted from the washed cell mass using a buffer with 1% sodium dodecyl sulfate, and purified by diafiltration using membrane filters and two-stage ion-exchange chromatography on hydroxyapatite. The protective antigen is isolated from the culture filtrate and purified by successive steps of concentration and diafiltration.

EFFECT: use of the invention enables to obtain in one processing chain the highly purified antigens of anthrax microbe - protective antigen and protein EA1 needed to create chemical vaccines.

3 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: alcohol (8-C) strain Saccharomyces cerevisiae No 8 having a high generative activity was deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number RNCIM B-3855 and can be used in production of alcohol.

EFFECT: invention enables to increase the alcohol yield and to reduce the formation of byproducts.

3 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of isolating and purifying recombinant human growth hormone which is secreted by Saccharomyces cerevisiae yeast during fermentation thereof in suitable conditions. The target protein is precipitated in biomass-free culture fluid by either acidification to pH 2.9-4.0 or adding polyethylene glycol with molecular weight of 3000-6000 Da. The obtained precipitate is then dissolved in a suitable solvent. Preliminary purification of the target protein is carried out either by anion-exchange chromatography at pH 5.6 or by diafiltration in the presence of 0.1-0.5 M sodium chloride. Main purification of the target protein is then carried out by anion-exchange chromatography at pH not below 7.3 and gel filtration.

EFFECT: invention enables to obtain a growth hormone which is free from parent proteins, host-producer protein and other impurities such as pigments, with output of up to 60%.

8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to use of the Lactobacillus paracasei CNCM I-2116 strain to treat irritable bowel syndrome. A probiotic includes dead Lactobacillus paracasei CNCM I-2116 bacteria, a fermentation substrate and/or material made from Lactobacillus paracasei CNCM I-2116.

EFFECT: invention provides the capacity to normalise post-infection hyper-contractible state of intestinal muscles.

2 cl, 4 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to Saccharomyces cerevisiae CNCM I-3856 and Saccharomyces cerevisiae var. boulardii CNCM I-3799 yeast strains that are used as probiotic which is suitable for preparing food or pharmaceutical compositions. Also disclosed is a composition which contains yeast strains Saccharomyces cerevisiae CNCM I-3856 and/or Saccharomyces cerevisiae var. boulardii CNCM I-3799 and/or at least one parietal mannoproteins EL 05 and EL 06 of the Saccharomyces cerevisiae CNCM I-3856 yeast strain.

EFFECT: invention enables to reduce relieve paint in the intestines, induction of anti-inflammatory action without pro-inflammatory action, difficult and reduced adhesion and population of the gastrointestinal tract with bacteria that are pathogenic and/or invasive in nature.

12 cl, 30 dwg, 6 tbl, 9 ex

FIELD: process engineering.

SUBSTANCE: invention relates to biochemistry. Effluents are cleaned of suspended substances, oil products, phenols and chlorides for water to be discharged into pool. Inner surface of filtration dam is processed by bacterial culture Pseudomonas fluorescens "ВКГ" RCAM 00538 with titre of 10-13-10-11 in amount of 30 mg/dm3 in dry weight to obtain biofilm. Said filtration dam is filled with water to be cleaned and kept therein for at least 3 days. Effluents are forced through filtration dam consisting of the following rocks: Crushed stone or sand-gravel mix, or mix of mudstone with siltstone.

EFFECT: efficient cleaning to MPC acceptable for water discharge into pool.

1 dwg, 14 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of altering immunomodulating properties of lipopolysaccharides of plague bacteria in vitro, which involves obtaining preparations of lipopolysaccharides (LPS) and mouse toxin (MT) Yersinia pestis with subsequent formation of a LPS-MT complex thereof. A modified form of LPS-MT is used as an inducer of synthesis of cytotoxins TNF-α and IFN-γ. To this end, a test sample is prepared, to which LPS is added in amount of 5 mcg (50 mcl from working dilution of 100 mcl/ml) and MT is added in amount of 50 ng (5 mcl from working dilution of 10 mcg/ml); the sample is then incubated for 30 min at 37°C. The volume of the sample in eppendorfs is then brought to 100 mcl with sterile buffered physiological solution of NaCl and the obtained mixture is added a tray dimple containing a culture of human monocyte cell line U-937 (1×106 cells in a dimple); the latter is cultured in a medium of PRMI 1640 with simultaneous double control. Further, 1, 4, 20 hours after the beginning of combined incubation of the preparations of LPS with monocytes, quantitative accounting of the synthesised cytotoxins is carried out, wherein change in the immunomodulating properties of LPS of plague bacteria in vitro is determined from the amount of cytotoxins produced and the dynamics of their synthesis.

EFFECT: invention enables to alter immunomodulating properties of lipopolysaccharides of plague bacteria in vitro, which enables to realise toxic potential of the endotoxin of plague bacteria.

2 cl, 8 dwg, 2 ex

FIELD: biotechnologies.

SUBSTANCE: strain Rhodococcus erythropolis 1-KP, extracted from podzol soil contaminated with oil from the territory of the Kolsky peninsula, having high speed of oil utilisation, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VNIISHM) under the number RCAM01142 and may be used for treatment of contaminated soils from oil.

EFFECT: improved quality of soil cleaning from oil.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: strain pseudomonas citronellolis 48-U, having high speed of oil and diesel fuel recycling, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VIISHM) under the registration number RCAM 01441 and may be used for treatment of contaminated soils from oil and diesel fuel.

EFFECT: improved quality of soil cleaning from oil and diesel fuel.

3 tbl

FIELD: biotechnologies.

SUBSTANCE: strain Rhodococcus fascians 4-G, extracted from soil contaminated with black oil and sampled from the territory of a boiler plant located in the settlement Gorelovo, Leningrad region, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VNIISHM) under the number RCAM01140 and may be used for treatment of contaminated soils from oil.

EFFECT: invention makes it possible to increase quality of soil treatment from oil.

3 tbl

FIELD: food industry.

SUBSTANCE: method envisages bacteria suspension production in a liquid nutrient medium, the bacteria separation from the suspension by way of centrifugation to produce a biomass, the produced biomass mixing with a protective medium containing sucrose and glycerine and the produced mixture freezing at a temperature of (-18)-(-20)°C. The liquid nutrient medium contains components at the following ratio, wt %: (10 - 12) - sucrose, (3 - 5) - malt sprouts and (4 - 6) - chalk. Before freezing the biomass mixture with the protective medium is cooled at a temperature of 4-6°C, maintained at the said temperature during 1 hour.

EFFECT: enhancement of biosynthetic activity of Lactobacillus delbrueckii lactic acid bacteria during preservation by freezing and subsequent storage.

1 tbl, 3 ex

FIELD: food industry.

SUBSTANCE: one proposes a composition for preparation of a food product using yeast, containing: instant active dry yeast particles, at least one biologically-compatible oil and/or at least one biologically-compatible paraffin where the weight ratio of the said oil and/or paraffin to instant active dry yeast is higher than 0.4:1; additionally, the composition optionally contains at least one biologically-compatible hydrophilous component. All the components are homogeneously dispersed; the dry substance content in the composition is higher than 90 wt %, and lower than 98 wt %. Additionally, one proposes a method for preparation of a food product using yeast, involving stages of mixing for homogeneous distribution of all the said components. The composition, according to the invention, is in a liquid, paste-like or powder form.

EFFECT: oil or paraffins presence in the composition protects yeast during storage and ensures good long-term stability of yeast enzyme capability under no special physical conditions of storage.

30 cl, 3 dwg, 33 tbl, 16 ex

FIELD: medicine.

SUBSTANCE: method involves vitrification of biological objects, preferentially gametes and embryos, in a flexible polycarbonate dropper for denudation and transfer of gametes and embryos and cumulus oocyte complexes of the diameter of 130-600 mcm and defrosting. A small amount of the vitrified solution free from biological objects is withdrawn in the dropper. Then the vitrified solution containing biological objects is withdrawn in the dropper, and the vitrified solution is added so than biological objects are at least at 10 mm from a distal end of the flexible polycarbonate dropper. The dropper is sealed from the distal end and checked for leaks by a Strippetor; a proximal end of the dropper is sealed. Then it is immersed in liquid nitrogen for min. 20 sec. and packed in a loaded outer casing culm of the min. volume of 0.25 ml for vertical orientation to be thereafter sealed. Defrosting is enabled by opening the outer casing culm, removing the sealed dropper with biological objects, placing the dropper in a waterbath at temperature 37 degrees Celsius. The dropper is opened from both sealed ends, and vitrified biological objects are tapped off into a defrosting solution with the use of a rubber bulb put on a wider portion of the dropper.

EFFECT: reduced price cost of the vitrification process and consumption of the frosting and defrosting solutions for biological objects.

6 dwg

FIELD: medicine.

SUBSTANCE: materials containing biologically active substances in a liquid phase are transformed in a micro-drop state stabilised by a dry high-disperse hydrophobic decoupler of nanosized particles to be dehydrated with using a sorbent at temperature minus 10-20°C.

EFFECT: invention allows higher activity of the active substances in process of dehydration of labile biologically active materials.

5 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: materials containing biologically active substances in liquid phase are converted to a microdroplet state stabilised with dry finely dispersed hydrophobic separator with nanosized particles, followed by dehydration, with weight ratio of the liquid phase to the sorbent from 1:4 to 1:8.

EFFECT: invention increases shelf life of active substances during storage of dehydrated labile biologically active materials.

1 dwg, 5 tbl, 12 ex

FIELD: food industry.

SUBSTANCE: method for production of the preparation of a partially dehydrated liquid bacteria inoculant involves: production of a liquid inoculant of bacteria grown basically till stationary phase with the bacteria represented by one or more genera of Rhizobium, Bradyrhizobium, Pseudomonas and Serratia and addition of a dehydrating additive including a desiccant to the liquid bacteria inoculant in an amount sufficient for partial dehydration of the liquid bacteria inoculant to produce the preparation of the partially dehydrated liquid bacteria inoculant; the desiccant is represent by one or more compounds selected from among trehalose, sucrose, glycerine, triethylene glycol and mannitol in an amount from approximately 5% to approximately 50% (wt/vol) of the preparation of the partially dehydrated liquid bacteria inoculant. Subsequent application of the produced onto a dry medium enables production of a dry granular bacteria inoculant.

EFFECT: invention allows to enhance bacteria survival ability and stability in inoculants, in packages and on seeds.

23 cl, 8 dwg, 5 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method provides preparation of a protective medium of phosphate buffer of pH 7.2 containing glycerin, lactose, sodium citrate, gelatin. The prepared protective medium is mixed with lactic bacteria in the ratio 1:1 and kept for 15-20 minutes at temperature 17 to 27°C to be agitated, granulated and frozen in liquid nitrogen.

EFFECT: method improvement.

2 ex

FIELD: medicine.

SUBSTANCE: method of obtaining composition including dried bacteria involves cultivation of one or more live bacteria types; mixing cultivated bacterium with one or more carriers; bacteria processing by impulse electromagnetic fields of 2 mV/cm at 50 Hz and 55 V; incubation of culture-carrier mix for at least 6 hours; and bacteria drying to reduce humidity level approximately to 1 to 6 wt %. Obtained composition can be applied for coating of seeds of other reproductive plant material, adding to plant growing medium, for sewage and/or chemical and biological waste treatment, contaminated soil treatment, for introduction of acceptable microbes to food and/or animal forage, for milk bacteria supply in medical purposes.

EFFECT: combination of mixing cultivated bacterium with one or more carriers and microbe processing in the composition enhances original survival rate and composition storage life.

22 cl, 3 dwg, 11 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: there is disclosed leprosy microbacteria storage method consisting in low temperature effect on the infected substance. The stored substance is cooled at temperature -18°C that allows using household freezers.

EFFECT: simplified method of storage of M leprae.

3 ex

FIELD: veterinary medicine.

SUBSTANCE: dry bacterial compound contains, at least for 10% on a weight basis of total compound, the dry bacterial concentrate having bacteria concentration of at least 1×108 CFU/g and water activity of less than 0.5, and stabiliser having water activity of less than 0.5 at water content of 10%. There also provided is packed bacterial compound, batched compound and stabilisation method of dry bacterial compound by mixing thereof with stabiliser having water activity of less than 0.5 at water content of 10%; at that, total bacterial compound has water activity of less than 0.5. Treatment method of the mammal requiring treatment involves introduction of batched compound according to the present invention.

EFFECT: improved stability of the product, and higher number of bacteria per the product dose.

26 cl, 1 dwg, 1 tbl, 14 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a polymyxin derivative having a total of three positive charges at physiological pH and where the terminal moiety (D) of the derivative contains a total of 1-5 carbon atoms; a method for production thereof; a composite product containing at least two such derivatives; a pharmaceutical composition containing a polymyxin derivative; a method of sensitising gram-negative bacteria to an antibacterial agent; a method of producing novel antibiotics.

EFFECT: invention also relates to use of the polymyxin derivative in producing a medicine for sensitising gram-negative bacteria to antibacterial agents.

23 cl, 8 tbl, 8 ex

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