JP4410992B2 - Bacterial strains, treated plant extracts, compositions containing them, methods for their preparation, and their therapeutic and industrial applications - Google Patents

Abstract

The present invention discloses: (i) a non-pathogenic probiotic microorganism and its probiotic/therapeutic uses; (ii) a formulation comprising an aqueous solution of a volatile fraction (VF) prepared from the extract of at least one plant derived material and its therapeutic uses; (iii) a process of manufacturing the formulation from the plant derived material; (iv) a probiotic composition comprising the non-pathogenic probiotic microorganism of the invention and/or other probiotic microorganism(s) and the formulation of the invention, and its probiotic/therapeutic uses; (v) a composition for industrial applications comprising the formulation of the invention and microorganism(s) of industrial applicability; and (vi) industrial processes and apparatuses in which the latter composition is used.

Description

Detailed Description of the Invention

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to (i) non-pathogenic probiotic microorganisms and their probiotic / therapeutic uses, (ii) volatility prepared from at least one plant-derived extract. A formulation comprising an aqueous solution of a sex fraction (VF) and therapeutic use thereof, (iii) a method for producing such a formulation from plant-derived materials, (iv) a non-pathogenic probiotic microorganism of the invention and / or Or probiotic compositions comprising other probiotic microorganisms and formulations according to the invention, and their probiotic / therapeutic uses, (v) industries comprising the formulations according to the invention and microorganisms having industrial utility And (vi) industrial processes and equipment in which the latter composition is used.

  Under normal conditions, the microbiota of the gastrointestinal (GI) tract contributes significantly to the health and well-being of the individual. It is well known that the microbiota is a complex and diverse microbial population that can have both beneficial and harmful effects on an individual. Under normal conditions, the fetus in the uterus is sterile, but when it passes through the vagina at birth, the fetus acquires microorganisms that result in the formation of gut microbiota. The final intrinsic gastrointestinal microbiota that stabilizes in the gastrointestinal tract is a very complex population of over 1000 microorganisms consisting of 400 different types of bacteria [Fuller R. et al. J. et al. Applied Bacteriology, 66: 365-378 (1989)]. The composition of the gut microbiota is determined by host and microbial factors. There are many bacteria that can survive and grow in the GI tract, but many bacteria cannot survive and grow in the GI tract. In addition, surviving organisms must avoid the habit of moving the bacteria away with food in normal cases. This can be achieved by the bacteria immobilizing themselves by binding to the digestive tract wall and / or by growing at a faster rate than the rate of movement by peristalsis.

  In general, the presence of gut microbiota is symbiotic. This is because the gut microbiota helps break down undigested food. The gastrointestinal microflora also protects individuals from infections caused by pathogens. This latter phenomenon is described by names such as “bacterial antagonism”, “bacterial interference”, “barrier effect”, “fixing resistance”, “competitive exclusion” and the like.

  This gastrointestinal protective microflora is very stable. However, this is not very effective for young people, elderly people and patients with reduced resistance. In addition, it can be affected by certain dietary and environmental factors. The three most important factors are excessive hygiene, antibiotic treatment and stress.

  In situations where the balance of the gastrointestinal microbiota is adversely affected, probiotics are potentially important in restoring the gastrointestinal microbiota and returning the individual host to normal.

  Probiotics are a class of microorganisms defined as living microorganisms that have a beneficial effect on animal and human hosts. Beneficial effects include improving the microbial balance of the gut microbiota, or improving the nature of the intrinsic microbiota. The beneficial effects of probiotics can be mediated by direct antagonistic effects on specific groups of organisms that cause a decrease in number, or by their effects on metabolism, or by stimulating immunity. Probiotics can reduce the number of undesired organisms by competing for nutrients or adhesion sites by producing antibacterial compounds. In addition, probiotics may alter microbial metabolism by increasing or decreasing enzyme activity, or may stimulate the immune system by increasing antibody levels or by increasing macrophage activity.

  International Patent Publication WO 95/16461 describes anaerobic bacterial probiotic compositions that are effective in suppressing or inhibiting Salmonella colonization of domesticated animals. The probiotic composition comprises a population or culture of 29 substantially biologically pure bacteria (particularly E. coli). However, the suppression of pathogens by the probiotic composition described in this PCT publication requires the combined action of numerous bacterial strains.

  International patent publication WO 97/35596 describes the administration of a freshly prepared probiotic mixture each time obtained by mixing a powder containing Lactobacillus reuteri, Lactobacillus acidophilus and Bifidobacterium infantis with a liquid. . This mixture has been described as effective in preventing infectious diarrhea or diarrhea caused by antibiotic treatment in humans. However, the lyophilized live bacteria are in an asphyxia state. In order to restore its vitality, the microorganisms must be moistened before administration. This is usually not preferred because many bacteria cannot tolerate rehydration. Moreover, surviving organisms are not immediately metabolically active and cannot survive the extreme acidic conditions of the stomach. Furthermore, when administered to a dosage form that develops diarrhea, the removal rate from the gastrointestinal tract is higher than the rate at which bacterial vitality is restored, resulting in much less or no beneficial effect.

  Conservation of viability and protection of activity by its formulation of probiotic organisms is the subject of numerous publications. In International Patent Publication WO 98/26787, a resident population of lactic acid-producing microorganisms (preferably lactic acid bacteria) in the GI tract of animals is optionally combined with prebiotic microorganisms and / or probiotic microorganisms. It is described that the enhanced β-glucan is given to lactic acid-producing microorganisms.

  International Patent Publication WO 97/34591 also contains selected modified or unmodified starches or mixtures thereof that act as carriers for one or more probiotic microorganisms and as a growth or maintenance medium for the microorganisms. Giving to an individual has been described as enhancing a resident population of microorganisms or suppressing an unwanted resident population at selected sites in the individual's GI tract. The probiotic component binds to the carrier in a manner that protects the microorganisms as it passes to the colon or other areas of the GI tract.

  Microorganisms are used in various industrial processes. These processes can be divided into continuous processes and batch processes. For example, in a continuous process used to produce a variety of fermentation products, including ethanol and methanol, such as an aerobic or anaerobic continuous fermentation production line, the microbial population in the production line starts the process. The fermentable raw material is continuously fed to the production line in a controlled manner so as to be maintained from end to end, and the fermentation product is continuously withdrawn from the production line. In batch processes, such as, but not limited to, batch aerobic or anaerobic fermentation, and biodegradation of oil, for example in oil spill accidents, the fermentable raw material is a microorganism (one Or mixed with multiple populations). In both continuous and batch fermentation processes, it is sometimes desirable to initiate or recover the process with a viable large population of the microorganisms (one or more) involved. For example, when biodegrading oil, for example, as large as possible for Pseudomonas spp. Or Alcaligenes spp. So that the time to fully biodegrade the oil is minimized. It is convenient to start the process with a microbial population. When using a microorganism-enriched filter to biodegrade organic gases or organic volatiles, the result is a continuous process in which contaminating compounds are broken down into harmless compounds. However, the microorganisms present in the filter rely on a continuous supply of organic gases or organic volatiles for their survival. Nevertheless, in some cases, the supply of organic gas or organic volatiles is interrupted, resulting in the loss of microorganisms. Under such circumstances, it is required in a short time to restore the effective viable amount of microorganisms in the filter so that the functional filter can be restored immediately.

  Accordingly, there is a widespread need for formulations that can maintain a variety of microorganisms, including probiotic microorganisms and microorganisms useful in various industrial applications, in a viable state and in a metabolically active form over an extended period of time It is recognized and it is very advantageous to have such a formulation.

  As the invention is put into practice, unexpectedly, normal GI flora of humans and various mammals and birds can be restored alone by a single species of non-pathogenic probiotic microorganisms derived from E. coli. It was found. Surprisingly, this microorganism, like other microorganisms, is viable metabolically active in formulations containing aqueous solutions of the volatile fractions (one or more) of various plant extracts. It has also been found that it can be stored in form for an extended period of time. Probiotic compositions comprising probiotic organisms suspended in such formulations have been found to be effective in the treatment and prevention of various gastrointestinal abnormalities. Moreover, such formulations have been unexpectedly found to be effective in themselves as mammals and birds as weight gain enhancers and as immunostimulants.

DISCLOSURE OF THE INVENTION According to one aspect of the present invention, E. coli strain BU-230-98 (ATCC Deposit No. 202226 (DSM 12799)) is provided.

  According to another aspect of the present invention, a probiotic comprising a viable E. coli strain BU-230-98 (ATCC Deposit No. 202226 (DSM 12799)) and a formulation for maintaining the viability of this E. coli strain A composition is provided. The probiotic composition of the present invention preferably further comprises at least one flavoring agent. Preferably, the formulation is a volatile of the plant extract prepared by steam distillation of a plant (eg beet, dill, parsley or grapefruit) extract under reduced pressure and preferably at a bath temperature not exceeding 38 ° C. Contains at least one of the fractions (VF).

  The probiotic composition of the present invention is used to prevent or treat gastrointestinal infections or gastrointestinal abnormalities and / or to maintain or restore normal gastrointestinal microflora and / or to prevent or treat diarrhea. And / or to prevent or treat gastrointestinal infections caused by enteric pathogens such as gram-negative or gram-positive bacteria and / or to prevent or treat gastrointestinal Salmonella infections and / or, for example, , C.I. difficile, Salmonella spp. Prevent or prevent infectious diarrhea caused by Shigella bacteria, Campylobacter bacteria, E. coli, Proteus bacteria, Pseudomonas bacteria or Clostridium bacteria, or chronic diarrhea, or diarrhea resulting from antibiotic therapy or radiation therapy or chemotherapy It can be identified to treat and / or normalize physiological activity of the gastrointestinal tract.

  According to yet another aspect of the invention, a formulation comprising at least one volatile fraction (VF) of a plant extract is provided. In this case, the volatile fraction is prepared by steam distillation of the plant extract under reduced pressure and preferably at a bath temperature not exceeding 38 ° C. The plant extract is preferably obtainable from a plant organ selected from the group consisting of leaves, stems, roots and fruits. Plants can include vegetables such as soybeans, alfalfa, garlic, beet and cabbage, or herbs such as parsley, mint and dill. The formulations of the present invention can further include beehive products such as but not limited to propolis. The formulations of the present invention can be used as animal feed additives or food additives to enhance animal weight gain in the dosage form. The formulations of the present invention can also serve to preserve viable bacteria, as further described herein.

  Thus, according to a further aspect of the present invention there is provided a method for preparing a volatile fraction of a plant. This method is obtained in (a) a step of pulverizing plant-derived material to obtain plant biomass, (b) a step of mixing plant biomass with water and stirring at ambient temperature, and (c) step (b). Steam-distilling the resulting mixture under reduced pressure and at a bath temperature not exceeding 38 ° C., and (d) collecting the volatile fraction obtained from steam distillation. Preferably, the reduced pressure is from 5 mbar to 10 mbar. This formulation can be used as a food additive, a feed additive and has unexpected therapeutic uses as well as favorable bacterial storage properties. A therapeutic formulation containing a volatile fraction of plants is also disclosed, which is physiologically or veterinarily acceptable for at least one of the volatile fractions (VF) of the plant extract. It can be prepared by mixing with additives or carriers or diluents. Preferably, the volatile fraction is prepared by steam distillation of the plant extract under reduced pressure and at a bath temperature not exceeding 38 ° C.

  Thus, according to yet another aspect of the present invention, (a) an effective amount of at least one viable probiotic microorganism having beneficial biological or therapeutic activity in the gastrointestinal tract; Probiotic compositions comprising at least one volatile fraction (VF) of a plant extract, preferably prepared as described herein, are provided. The probiotic microorganism is Escherichia coli, preferably BU-230-98 strain (ATCC Deposit No. 202226). This can be identified to treat or prevent any of the various abnormalities / pathologies / diseases / syndromes described above, and in addition or alternatively, to treat or prevent dyspepsia symptoms, eg, immune It can be identified to enhance the immune response in patients suffering from immune abnormalities resulting from response suppression therapy.

  According to still another aspect of the present invention, (a) Escherichia spp., Alcaligenes spp., Arthrobacter spp., Bifidobacterium spp.), Lactobacillus spp., Lactococcus spp., Nitrosomonas spp. and Pseudomonas spp. (B) the viability of said at least one microorganism at room temperature for at least 2 months, preferably at least 3 months to 6 months More preferably, a composition comprising at least one volatile fraction (VF) of a plant extract selected to last for 6 to 12 months or longer.

  According to another aspect of the present invention, a method of growing E. coli strain BU-230-98 (ATCC Deposit No. 202226 (DSM 12799)) is provided. This method involves inoculating a growth medium with a starter culture of E. coli strain BU-230-98 (ATCC Deposit No. 202226 (DSM 12799)) and growing the E. coli strain until the optical density at 650 nm is 15-30. Including the step of

  According to yet another aspect of the invention, a method for producing a probiotic composition is provided. This method involves suspending a viable E. coli strain BU-230-98 (ATCC Deposit No. 202226 (DSM 12799)) in a formulation to maintain the viability of this E. coli strain. Preferably, the formulation is from any plant organ selected from the group consisting of leaves, stems, roots and fruits, for example any vegetable and herb such as soybean, parsley, mint, dill, alfalfa, garlic, beet or cabbage. It comprises at least one volatile fraction (VF) of the plant extract, preferably prepared by steam distillation of the resulting plant extract under reduced pressure and a bath temperature not exceeding 38 ° C.

  According to yet another aspect of the invention, a method of preparing a probiotic composition is provided. This method involves the effective amount of at least one viable probiotic microorganism having beneficial biological or therapeutic activity in the gastrointestinal tract, at least one of the volatile fractions (VF) of the plant extract. Suspending in a formulation containing

  According to yet another aspect of the invention, a method of preparing a composition comprising a viable microorganism is provided. The method comprises at least one formulation comprising at least one volatile fraction (VF) of a plant extract, selected to maintain the viability of at least one microorganism at room temperature for at least 2 months. Suspending the microorganisms. The microorganism can be, for example, an Escherichia coli bacterium, an Alkaligenes bacterium, an Arthrobacter bacterium, a Bifidobacterium genus, a Lactobacillus genus, a Lactococcus genus, a Nitrosomonas genus or a Pseudomonas genus.

  Further, according to a further aspect of the present invention, there is provided a microbial dispenser comprising a reservoir and a dispensing mechanism connected to the reservoir. In this case, the reservoir is in a formulation containing at least one volatile fraction (VF) of the plant extract, selected to maintain the viability of at least one microorganism for at least 2 months at room temperature. Contains at least one microorganism.

  Furthermore, according to a further aspect of the present invention, a biological biocontrol method is provided. The method includes the step of spraying at least one viable microorganism capable of biocontrolling an organism in an endemic area, wherein the at least one viable microorganism is the viability of at least one microorganism. Present in a formulation containing at least one volatile fraction (VF) of the plant extract, selected to persist at room temperature for at least 2 months.

  According to a further aspect of the present invention, a method for removing oil contamination is provided. The method includes the step of spraying at least one viable microorganism capable of degrading oil in a contaminated area, wherein the at least one viable microbe increases the viability of the at least one microbe at room temperature. Present in a formulation containing at least one volatile fraction (VF) of the plant extract, selected to last for at least 2 months.

  Furthermore, according to a further aspect of the present invention, there is provided a method of reviving an organic gas decomposing microbial population in a biofilter, the method applying to the filter at least one viable microorganism capable of decomposing organic gas. Where the at least one viable microorganism is selected to maintain the viability of the at least one microorganism for at least 2 months at room temperature, the volatile fraction (VF) of the plant extract Present in formulations containing at least one of

  Furthermore, according to a further aspect of the present invention, a method for preparing a starter product for a fermentation process is provided. This method grows a sufficient amount of starter microorganisms and the starter microorganisms are selected to maintain the viability of the starter microorganisms at room temperature for at least two months (VF) Suspending in a formulation containing at least one of

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is described herein by way of example only with reference to the accompanying drawings. Referring now more particularly to the drawings in detail, the specific matter shown is by way of example only and for exemplary discussion of the preferred embodiments of the present invention, and the principles and concepts of the present invention. The emphasis is on being presented to provide what is considered to be the most useful and easily understood description of this aspect. In this context, no structural details of the invention have been given in more detail than is necessary for a basic understanding of the invention. It will be apparent, however, to one skilled in the art how some aspects of the present invention may actually be embodied by the description as understood in conjunction with the drawings.
FIG. 1 is a schematic view of a dispenser according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a volatile fraction prepared from (i) a non-pathogenic probiotic microorganism and its probiotic / therapeutic use, (ii) at least one plant-derived extract. A formulation comprising an aqueous solution of (VF) and its therapeutic use, (iii) a method of producing such a formulation from plant origin, (iv) a non-pathogenic probiotic microorganism of the invention and / or other Probiotic compositions comprising probiotic microorganisms and formulations according to the invention, and their probiotic / therapeutic uses, (v) industrial applications comprising the formulations according to the invention and microorganisms having industrial utility And (vi) industrial processes and equipment in which the latter composition is used.

  The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

  Before describing at least one embodiment of the present invention in detail, it should be understood that the present invention is not limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced in various ways or being carried out in various ways. It should also be understood that the expressions and terms used herein are illustrative and should not be regarded as limiting.

  In one aspect, the invention has beneficial physiological and / or therapeutic activity in the GI tract and is deposited with the ATCC under deposit number 202226 and deposited with the DSMZ under deposit number 12799. The present invention relates to a probiotic microorganism which is a non-pathogenic bacterium derived from Escherichia coli.

  “Probiotic” is used herein to describe an isolated bacterium that has the property of inhibiting the growth of at least one pathogen. As used herein, the inhibition test is a solid medium culture where the culture supernatant of an isolated candidate bacterium is observed for its property of inhibiting pathogen growth when added to the surface of the solid medium. It was an in vitro test. Typically, a paper disc impregnated with the culture supernatant of a candidate probiotic strain was placed on the surface of an agar plate seeded with the pathogen. The supernatant of the probiotic bacteria produced a clear agar ring or a reduced growth density ring showing pathogen inhibition in the vicinity of the disc. Various other tests for inhibition can be utilized or devised, including direct in vitro or in vivo that can result in a probiotic bacterial population similar to the probiotic bacterial population described herein. A typical growth competition test is included. Bacterial strains identified by any such test are included in the category of probiotic bacteria, as that term is used herein.

  The probiotic bacterium of the present invention can be used as a food additive or a feed additive, and as a result, supplemented with a viable E. coli strain BU-230-98 (ATCC Deposit No. 202226 (DSM 12799)) A food product or a feed product can be obtained. The food product of the present invention can further comprise at least one milk-derived substance and can be selected from the group consisting of cheese and yogurt.

  The invention further relates to a formulation comprising at least one volatile fraction (VF) of a plant extract. This volatile fraction is prepared by steam distillation of the plant extract under reduced pressure and bath temperature not exceeding 38 ° C.

  The plant body from which the volatile fraction can be obtained can be any suitable plant part such as fruit, leaf or root. Many plants are suitable as sources of such volatile fractions, such as apples, citrus fruits, soybeans, beets, cabbage, garlic and alfalfa, and herbs such as parsley, mint and dill. The formulations of the present invention can optionally further include an appropriate amount of a volatile fraction of a beekeeping product such as honey, propolis or other beehive product. This volatile fraction can also be prepared in the same manner as described herein to obtain a volatile fraction from a plant extract.

  The formulation itself can be used as a food / feed additive, as described in more detail in the examples below. It has been found by the inventors that this formulation has weight gain enhancing activity and constitutes one aspect of the present invention. According to one particular embodiment of this aspect of the invention, the animal weight gain formulation or feed additive is in a weight ratio of 2: 8: 1: 4 to distilled water, preferably the volatile fraction. Alfalfa, soybean, beet and dill volatile fractions. Alternatively, the formulation or feed additive of the present invention comprises distilled water and a volatile fraction of carrot, beet, dill and grapefruit, one part each for 100 parts water. Additionally or alternatively, the formulation or food additive of the present invention comprises distilled water and a volatile fraction of beet, dill and parsley, one part each for 100 parts water.

  In a further aspect, the present invention has the above formulation of the present invention and at least one viable microorganism, ie, therapeutic activity or beneficial biological activity in the GI tract of, for example, humans and / or animals. , Thereby to a composition comprising either a probiotic organism resulting in a probiotic composition, or an organism having an industrial use, thereby providing a composition having industrial utility.

  The particular advantage of the probiotic and industrial compositions of the present invention stems from the fact that the composition is a liquid preparation. Because it is in a biologically active state, the formulation of the present invention also serves as a support medium for live bacteria, as opposed to a lyophilized formulation in which the bacteria are in a mortal state. As a result, the probiotic composition of the present invention becomes active immediately after, for example, oral administration. This begins in the upper part of the GI tract where the primary effects of most enteropathogens that develop harmful gastrointestinal symptoms occur. The probiotic composition of the present invention can also be used as a weight gain preparation or food / feed additive. Similarly, the industrial compositions of the present invention can also be used to efficiently reverse microbial populations in industrial utility.

  Accordingly, probiotic bacteria other than such bacterial strains belonging to strain BU-230-98 (ATCC Deposit No. 202226, which is also deposited with DSM No. 12799) for probiotic applications Note that it can be used with a formulation derived from the plant material of the present invention. Such bacteria have a very broad spectrum of action. Such bacteria belong to the same phylogenetic group of most enteric pathogens and share the same survival system. Thus, suppression and elimination of enteric pathogens can include many different mechanisms, including, for example, antagonist secretion, competition for nutrient utilization, and competition for adhesion receptors. Thus, any non-pathogenic bacterium that meets these criteria can be used in the probiotic compositions of the invention.

  According to a further aspect of the present invention, the probiotic composition of the present invention can be used for the prevention or treatment of gastrointestinal infections. The term “gastrointestinal infection” shall be taken to mean any infection caused by enteric pathogens, including among others gram negative and gram positive bacteria. By improving the overall balance and health of the GI tract, the formulations and probiotic compositions of the present invention may also help prevent GI infections caused by yeasts, viruses and protozoa.

  The term “therapeutically effective amount” or “effective amount” is such an amount as determined by consideration as is known in the art for purposes herein. This amount must be sufficient to allow efficient recovery of the GI microbiota and thus provide normalization of GI tract function.

  In certain embodiments, a probiotic composition for preventing the development of gastrointestinal infections is described herein, wherein the volume ratio is, for example, 5: 1: 5: 15: 1 with distilled water. A formulation of the present invention made from volatile fractions of alfalfa, soybean, beet, dill and mint, and any probiotic bacterium such as E. coli ATCC Deposit No. 202226 (identical to DSM 12799) or any Includes therapeutically effective amounts of other probiotic bacteria.

  A specific example for gastrointestinal infections is infections caused by Salmonella bacteria, and the present invention is particularly advantageous in preventing or treating gastrointestinal infections caused by them.

  Further, the food additive or formulation of the present invention can be used alone or in combination with an effective amount of a probiotic microorganism such as E. coli strain ATCC Deposit No. 202226 (identical to DSM 12799). Can be used for the treatment or prevention of chronic diarrhea, or diarrhea caused by antibiotic treatment or chemotherapy.

  According to a further specific embodiment of the invention, such a probiotic composition for treating infectious diarrhea comprises distilled water and alfalfa with a volume ratio of eg 5: 1: 5: 15: 1, Soy, beet, dill and mint volatile fractions and a therapeutically effective amount of ATCC 202226 (DSMZ 12799) can be included.

  Infectious diarrhea can be caused by a number of factors such as C.I. difficile, Salmonella spp. It can be caused by a microorganism selected from Shigella, Campylobacter bacteria, E. coli, Proteus bacteria, Pseudomonas bacteria, Clostridium bacteria, Enterococcus staphylococci. These are just a few of the many infectious agents.

  Still further, the probiotic compositions of the present invention can be used to effectively restore the GI microbiota in a subject in need of such treatment that normalizes GI tract function. Such a composition is, for example, of alfalfa, soy, beet, dill, mint, parsley and cabbage with a ratio of distilled water and preferably a volatile fraction of eg 1: 5: 5: 2: 2: 1. Each volatile fraction and a therapeutically effective amount of the probiotic bacterium ATCC Deposit No. 202226 (DSM 12799) can be included.

  For example, other objectives for the probiotic compositions of the present invention comprising at least one probiotic bacterium having a therapeutic action in the GI tract include alleviation of lactose intolerance in subjects suffering from lactose intolerance, total Immune system stimulation in subjects suffering from immune system abnormalities, which can be immune disorders caused by treatment of enteritis, treatment of constipation, reduction of blood cholesterol levels, treatment of dyspepsia, and / or immunosuppressive treatment Is included.

  In a different aspect, the present invention relates to a method for preparing a volatile fraction of a plant extract. This method includes the following steps: (a) pulverizing plant-derived material to obtain plant biomass; (b) plant biomass obtained in step (a) into plant biomass having a weight ratio of 1 part. Mixing with water, which is 3 parts water, and stirring the mixture at ambient temperature for at least 2 hours; (c) bath temperature not exceeding 38 ° C. under reduced pressure and not exceeding 38 ° C. And (d) collecting the volatile fraction obtained from the steam distillation, which can be further diluted with a suitable buffer solution.

  The volatile fraction of the present invention can be mixed with water to obtain the food / feed additive or formulation of the present invention. The food / feed additive or formulation of the present invention can also be prepared by mixing two or more plant volatile fractions obtained by the method of the present invention. This mixture can be further mixed with water.

  The volatile fraction can be prepared from any suitable fruit, vegetable, leaf, stem or root of the plant. The plant can be, for example, an apple, citrus fruit, soybean, beet, garlic, cabbage or alfalfa, or a herb such as parsley, mint or dill. It should be noted that where appropriate, the formulation may further comprise a volatile fraction derived from apiculture products such as honey or propolis or other beehive products. These volatile fractions can be prepared in the same manner as the plant extract volatile fraction.

  According to the method of the invention, the distillation step is preferably carried out under reduced pressure of 5 mbar to 10 mbar.

  As indicated hereinabove, the food / feed additive or formulation of the present invention comprises at least one probiotic or industrial to obtain the probiotic or industrial composition of the present invention. It can be further combined with factors. Therefore, the preparation method of the invention also provides at least one live probiotic or industrial microorganism in the volatile fraction obtained in step (d) or as described above. It may further comprise the step of suspending in a mixture of such volatile fractions.

  According to a particular method of the present invention, the suspended probiotic microorganism is derived from E. coli and deposited with the ATCC under deposit number 202226 (and with the DSM under deposit number 12799). Can be a simple bacterium.

  Finally, the present invention relates to the use of the volatile fraction (VF) of plant extracts in the preparation of food / feed additives. In this case, VF is prepared by steam distillation of the plant extract at a bath temperature not exceeding 38 ° C. or by steam distillation from a beekeeping product extract such as honey or propolis.

  A further aspect of the invention includes a method for preparing a composition comprising viable microorganisms. This method is accomplished by suspending at least one microorganism in a formulation containing at least one volatile fraction (VF) of the plant extract. The formulation is selected to maintain the viability of the microorganism (one or more) for at least 2 months at room temperature. The microorganism chosen can be probiotic or have industrial utility. Organisms that can be maintained in the formulations of the present invention include Escherichia spp., Alcaligenes spp., Arthrobacter spp., Bifidobacterium. spp.), Lactobacillus spp., Lactococcus spp., Nitrosomonas spp. and Pseudomonas spp.

  As shown in FIG. 1, in another aspect, the present invention provides a microbial dispenser 10. The dispenser 10 includes a reservoir 12 and a dispensing mechanism 14 connected thereto. Reservoir 12 contains at least one microorganism in a formulation containing at least one volatile fraction (VF) of the plant extract. The formulation is selected to maintain the viability of the microorganism (one or more) for at least 2 months at room temperature. The reservoir 12 can be refrigerated to, for example, about 2 ° C. to 20 ° C., preferably about 4 ° C. The dispenser 10 can be an air floating dispenser so as to allow the dispersal of the pest control bioagent. The dispenser can be handheld. In any case, the dispenser can be designed such that a predetermined volume is automatically dispensed from the dispenser, for example on a regular basis. For this purpose, the dispenser 10 can include a programmable control mechanism 16 or a preset control mechanism 16, a valve 18, and the like.

  Thus, according to another aspect, the present invention provides a method for biocontrol of an organism achieved by spraying at least one viable microorganism capable of biocontrolling the organism in an endemic area. The viable microorganism (one or more) is at least a volatile fraction (VF) of the plant extract selected to maintain the viability of the at least one microorganism for at least 2 months at room temperature. Present in formulations containing one.

  Still further, according to another aspect, the present invention provides a method for removing oil contamination, achieved by spraying at least one viable microorganism (such as Pseudomonas bacteria) capable of degrading oil in a contaminated area. I will provide a. Viable microorganisms (one or more) are selected to maintain the viability of said at least one microorganism at room temperature for at least 2 months, the volatile fraction (VF) of the plant extract Present in formulations containing at least one of

  Furthermore, according to another aspect, the present invention provides a filter with at least one viable microorganism (such as Pseudomonas, Nitrosomonas or Arthrobacter) capable of degrading organic gases, A method is provided for reviving an organic gas degrading microbial population in a biofilter. Viable microorganisms (one or more) are selected to maintain the viability of said at least one microorganism at room temperature for at least 2 months, the volatile fraction (VF) of the plant extract Present in formulations containing at least one of

  Still further, according to another aspect of the present invention, a sufficient amount of a starter microorganism (such as a Lactococcus bacterium, an Escherichia bacterium, a Bifidobacterium bacterium, a Lactobacillus bacterium, or a Pseudomonas bacterium) is grown, and The starter microorganism is suspended in a formulation containing at least one volatile fraction (VF) of the plant extract selected to maintain the viability of the starter microorganism at room temperature for at least 2 months. This provides a method for preparing a starter product for a fermentation process.

  Further objects and advantages and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Further, each of the various embodiments and aspects of the invention as set forth hereinabove and described in the claims section below is found experimentally in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above description, illustrate the invention in a non-limiting manner.

Example 1
Preparation of “volatile fraction” of extracts from plant extracts or beekeeping products Fresh vegetables obtained from the market were thoroughly washed with tap water, chopped and finely ground in an industrial blender. Distilled water was added to this vegetable biomass at a ratio of 3 parts water to 1 part vegetable biomass by weight and left under stirring for at least 2 hours at ambient temperature.

  The mixture is then transferred to a rotary evaporator evaporation flask under conditions such that the temperature of the water bath does not exceed 38 ° C. and the temperature at the condenser inlet is 2 ° C. to 5 ° C. under reduced pressure (5-10 mbar). And evaporated. Approximately 1 liter of condensate (volatile fraction) was recovered per 2.5 kg of plant biomass. This material can be stored refrigerated for at least 12 months without losing its properties, as further detailed in the present invention.

  The same procedure can be performed replacing plant material with beekeeping products such as honey or propolis.

  Usually, each “volatile fraction” was prepared from one kind of vegetable and used at different dilutions to prepare different mixtures for different purposes.

Example 2
Isolation and propagation of probiotic organism E. coli ATCC Deposit No. 202226 Long-term (2 months) stored above 37 ° C. in a formulation containing a volatile fraction of plant-derived material as described in Example 1 The probiotic organism E. coli (ATCC No. 202226 in 1999) is transferred by sequentially transferring isolates selected as isolates capable of competing with pathogens as detailed below. On March 3, and deposited with DSM No. 12799 on May 4, 1999) was isolated from E. coli M-17. The probiotic organism E. coli (deposited at ATCC No. 202226 on May 3, 1999 and DSM No. 12799 on May 4, 1999) was grown in the following medium (g / l): : (NH ₄ ) ₂ SO ₄ 5 g / l, KH ₂ PO ₄ 13 g / l, Na ₂ HPO ₄ 13 g / l, MgCl ₂ 3 g / l, CaCl ₂ 0.3 g / l, yeast extract 10 g / l, soybean Peptone 10 g / l, glucose 5 g / l. Additional nutrients (yeast extract 1 g / l, soy peptone 2.5 g / l, glucose 90 g / l) according to the growth of the culture in such a way that the glucose concentration in the fermentation broth is kept at a level of 2 g / l Added continuously. The pH of the fermentation broth was kept neutral by continuously adding 4N NH ₄ OH. Incubation was carried out at 30 ° C. for 16 hours with aeration of 0.5 vvm in a standard fermenter. After 16 hours, the growth became confluent. This procedure yielded 10 ¹⁰ to 10 ¹¹ cells / ml. E. coli cells were collected by centrifugation, resuspended in saline and precipitated again. This microbial biomass can be stored in physiological saline for 48 hours in a refrigerator without loss of viability.

Example 3
Preparation of food / feed additives to promote weight gain in mammals and birds The mixture contained the following volatile fractions: alfalfa 50 ml / l, soybean 200 ml / l, prepared as described in Example 1. Beet 25ml / l and dill 100ml / l. The remaining amount consisted of distilled water.

Example 4
Preparation of food supplement to normalize the function of the human GI tract The mixture contained the following volatile fractions: alfalfa 50 ml / l, soybean 10 ml / l, beet 50 ml, prepared as described in Example 1. / l, dill 50 ml / l, mint 20 ml / l, parsley 20 ml / l, and cabbage 10 ml / l. The remaining amount consisted of distilled water. E. coli (ATCC 202226) cells (Example 2) were suspended in the mixture at a cell concentration of 10 ⁷ cells / ml. NaCl (4-10 g / l) may be added as needed to improve the taste.

Example 5
Preparation of feed additive formulation to prevent GI infections in mammals and birds The mixture contained the following volatile fractions: alfalfa 50 ml / l, soybean 10 ml / l, prepared as described in Example 1 , Beet 50 ml / l, dill 150 ml / l, and mint 10 ml / l. The remaining amount consisted of distilled water. E. coli (ATCC 202226) cells (Example 2) were suspended in the mixture at a cell concentration of 10 ⁷ cells / ml. NaCl (4-10 g / l) may be added as needed to improve the taste.

Example 6
Antagonistic activity of E. coli (ATCC 202226) against Salmonella typhimurium (ATCC 14028) in Petri dishes containing modified brilliant green agar (selective growth medium for Salmonella bacteria). Inoculated with typhimirium. Wells with a diameter of 9 mm were prepared in agar. A fixed volume of food supplement (Example 4) was placed in each well and the plates were incubated at 35 ° C. for 24 hours. The same was repeated, but instead of the food supplement, the liquid obtained from its filtration through a microbiological filter membrane (0.45 μm pore size) was placed in the well.

  Around each well containing the feed additive is S. cerevisiae. A zone of inhibition (10-17 mm) in which no typhimirium colonies were present was observed. There was no zone of inhibition around the well containing the filtrate without the probiotic organism.

Example 7
Shigella sps. E. coli ATCC 202226 and its parental strain E. coli M-17 flexneri, S.M. Sonnei, E. coli (ATCC 202226) and M-17 cultures were grown separately at 37 ° C. for 18-20 hours in normal nutrient agar. All cultures were collected in saline and diluted to an optical density of 10 Klett units. A portion (1 ml) of a Shigella species dilution culture, alone or in combination with a dilution culture (1 ml) of E. coli (ATCC 202226), into an aerated cap test tube containing normal nutrient medium (5 ml). Vaccinated. Tubes were incubated at 37 ° C. for 24 hours. Pathogen and colony forming unit (CFU) numbers of E. coli (ATCC 202226) were determined by seeding the cultures on common nutrient agar. Table 1 shows the CFU numbers of two Shigella species in a pure culture and a mixed culture with two probiotic E. coli species.

Example 8
Application of food supplement (Example 4) and colibacterin (dried formulation of E. coli M-17) in hospitalized patients with gastroenteritis A group of 60 patients who developed gastroenteritis with hospitalization were treated by sex, age and gastrointestinal inflammation status. Randomly divided into three small groups with similar distributions of severity. All patients received normal supportive care including rehydration and vitamins. The severity of the condition did not require antibiotic treatment. A first group of 20 patients received 10 ml of food supplement (Example 4) three times a day for 7 days, 30 minutes before meals. The same number of second groups were given colibacterin siccum as recommended by the manufacturer (one dose, 30 minutes before meal, twice a day). The third group received no supplements.
The onset of normalization of gastroenteritis symptoms (days) in all groups was recorded and is shown in Table 2 below.

  Colibacterin siccum is available from BIOMED Ltd. (Moscow, Russia) is a lyophilized preparation of live E. coli M-17 and is recommended for use in diarrhea [Vidal Handbook: Pharmaceutical preparations in Russia (NB Nicolaeva, BP Alperovich) And V.N. Sovinov), AstraPharmService, 1997, Moscow, 275].

Example 9
Application of food supplement (Example 4) in patients with acute GI infection Patients with severe GI infections of various etiologies (salmonellosis, colibacillosis, bacterial dysentery, staphylococcal infections and food poisoning of unknown etiology) It was included in this study. All patients were admitted to hospital with acute onset of gastroenteritis disease and aspect. A total of 186 patients were treated with food supplements, and a similar group of 102 patients received standard treatment.
The onset of normalization of gastroenteritis symptoms in all groups was recorded (in days) and is shown in Table 3 below.

  In another study, a group of 30 patients with typhoid fever was treated with food supplements. In 80% of patients, disease symptoms disappeared within 3 days. In only 3 cases, the adjunct treatment was discontinued due to the development of more important chronic colitis.

Example 10
Application of food supplement (Example 4) in patients with GI abnormalities caused by antibiotics Patients with severe GI abnormalities were divided into two groups. Group I includes H. Forty-eight patients with peptic ulcer disease who developed GI abnormalities after antibiotic treatment for Pylori were included. Group II included 22 patients who developed GI abnormalities after antibiotic treatment of pneumonia.
Food supplement (5 ml) was administered three times a day for 7 days before meals. In both groups, diarrhea symptoms disappeared in 2 to 3 days in all patients. After supplementation, complete normalization of the gut microbiota was observed in 84.5% of patients. Its normalization is a dramatic increase in lactic acid bacteria and Bifidobacterium, a decrease in the overall count of E. coli, hemolytic E. coli and other pathogens (such as Staphylococcus, Proteus vulgaris and even Candida sps. ) Revealed by complete disappearance. In the remaining 15.5% of patients, a marked improvement was observed.

Example 11
Application of food supplement (Example 4) in patients with late stage radiation enteritis Food supplement (10 ml, 3 times a day for 14 days, 30 minutes before meal) after radiation therapy or combination of radiation and chemotherapy Twenty-four patients with uterine cancer, colon cancer and gastric cancer who developed pancreatitis were given.
An imaging material is provided wherein the weight ratio is> 0.1.

  Prior to using food supplements, all patients complained of frequent painful bowel movements, liquid stools (4-12 times a day). Mucosa (9 cases) and blood (3 cases) were seen in the stool.

Two or three days after taking the supplement, all patients had reduced pain and decreased stool frequency; the stool had a normal appearance. After 4 to 5 days, diarrhea symptoms disappeared and blood and mucous membranes were not seen. Blood analysis showed a strong improvement in blood indicators.
Symptoms persisted in a control group of similar size.

Example 12
Application of food supplement in AIDS patients (Example 4) Patients with AIDS frequently develop chronic diarrhea. Such a group of patients received 10 ml of food supplement (Example 4) three times a day for 20 days, 30 minutes before meals. No supplement was given to the control group. The results of treatment are shown in Table 4 below.

  Patients who have been given food supplements normalize the gut microbiota: a decrease in the overall number of coliforms, disappearance of hemolytic E. coli, an increase in the number of lactobacilli and bifidobacteria, Candida sps. Decreased.

Example 13
Application of food / feed additive (Example 3) to accelerated weight gain in healthy piglets Healthy piglets were orally dosed with 3 ml of food / feed additive per day until weaning. The group to which the feed additive was administered gained an average body weight of 1.0 kg per head at the time of weaning than the control group.

Example 14
Application of food / feed additive in healthy piglets (Example 5) Hundreds of healthy piglets were orally given 3 ml of feed additive per head on the first and third days after delivery and at weaning Administered. Mortality was reduced by 50% compared to the control group that received standard preventive treatment with antibiotics. The feed additive administration group gained an average weight of 0.39 kg per head at the time of weaning than the control group. When dietary supplement treatment and antibiotic treatment were compared in the same litter, the weight gain in piglets containing the dietary additive was found to be 2.4 kg greater than the control.

Example 15
Application of feed additive (Example 5) in piglets showing diarrhea Several hundred piglets showing diarrhea were orally dosed daily with 5 ml of feed additive per head. A control group of the same size was treated with antibiotics (advosin, gentiamycin, amoxiline). Diarrhea symptoms in the feed additive administration group disappeared in 1 to 2 days. There was no death and the piglet developed normally. Antibiotics stopped diarrhea in the majority of piglets, but piglets remained impeded in their development.

  Antibiotic treatment for diarrhea was given over a week and feed additives were given over a period of 3 days to about 70 retarded piglets, generally considered a loss. All but two survived.

Example 16
Application of feed additive in healthy calves (Example 5) Healthy calves aged 5 days were administered 5 ml of feed additive in milk for 7 days per day. More than 95% of calves did not develop diarrhea until 14 days of age when a few positive cases for rotavirus were diagnosed. The incidence of diarrhea in this farm is usually 20-30%.

Example 17
Application of feed additive (Example 5) in calves exhibiting diarrhea A calf that develops diarrhea was administered daily with 10 ml of feed additive with milk for 3-5 days. Diarrhea symptoms disappeared in 1 to 2 days in 90% to 95% of calves. In the remaining 5% to 10%, diarrhea was caused by the virus. These calves were treated with antibiotics, but the results were poor.

Example 18
Application of feed additive (Example 5) in healthy lambs and lambs 1 day old healthy lambs and lambs were dosed with 3 ml of feed additive per day in milk for 7 days. In some cases, mild to normal signs of diarrhea were observed. These signs usually disappeared spontaneously or were treated without problems with increased feed additives (5 ml). The incidence of diarrhea in these farms is usually 50% in lambs and goats.

Example 19
Application of Feed Additive (Example 5) in Lambs and Lambs Showing Diarrhea The feed additive was tested in a livestock population suffering from pathogenic E. coli infection. Approximately 90-120 lambs and goats died from diarrhea the year before the experiment. Treatment with antibiotics was not effective as the disease suddenly developed and resulted in early death. Lambs and goats that develop diarrhea were daily administered with 5 ml of feed additive with milk for 3-5 days. Comparable groups of animals were treated with antibiotics as usually recommended. Symptoms of diarrhea disappeared in 1-2 days in about 90% of lambs and goats that received the feed additive. Their further development appeared normal. The control group that received antibiotics (gentamicin) showed much worse results. Diarrhea in this group persisted and repeated treatment with antibiotics was required. The growth of the control group was severely delayed.

Example 20
Application of feed additive (Example 5) in poultry Feed additive is added to drinking water during breeding period (42 days to 49 days) with an average intake of 0.01 ml additive per chick per day It was. A 3.2% increase in weight gain was observed in a controlled trial in broilers with a 4% improvement in food conversion.
Excellent results were also obtained in the case of turkeys 1 day to 6 weeks old. Each bird received 0.01 ml of additive per day. Over 10% weight gain was observed with reduced mortality. Birds still showing diarrhea were treated with 0.1 ml food additive per day without any antibiotic treatment. Better recovery from control birds treated with antibiotics alone was shown.

Example 21
Application of feed additive in dogs and cats (Example 5) By applying the feed additive in puppies, diarrhea symptoms stopped within 24 to 48 hours.

Example 22
Application of feed additive (Example 5) in mice; evidence for immune stimulation Two week old C57B1 mice were divided into four groups of 10 animals each. Mice were not given water from 4:00 pm to 8:00 am, then were given either 15 ml of water (Group 1 & Group 3) or feed additive (Group 2 & Group 4) . This treatment was repeated for 2 weeks. When this treatment was completed, the mice in groups 3 and 4 were subjected to laparotomy under ether anesthesia. A 1.5 cm incision was made in the abdominal wall. Intestinal irritation was performed with dry gauze. All mice were sacrificed 24 hours after laparotomy and the spleen was excised. Spleen cells were isolated and cultured for 48-72 hours in RPMI medium in the presence of phytohemagglutinin (PHA). In the group treated with the feed additive, the proliferative activity of spleen cells decreased by laparotomy was restored.
1. Spleen cell proliferation induced by PHA Control group / water 3060 ± 290
2. Control group / water / laparotomy 2120 ± 300
3. Test group / Bio cocktail 2740 ± 370
4). Test group / bio cocktail / laparotomy 3040 ± 520

Example 23
Application of food additive (Example 4) in humans; evidence for immune stimulation Many patients have been operated on for uterine, cervical, gastric and intestinal malignancies. Gynecological patients (20 patients) received subsequent radiotherapy, and abdominal patients (30 patients) received chemotherapy. About half of the patients were given food additives twice daily during the treatment period. Significant immune stimulation was observed in all patients given food additives, as shown below:

Example 24
Bacterial preservation effect of the formulation E. coli cells were dispersed in the formulation prepared as described in Example 1 above and kept at room temperature for 6 months. At both the beginning and end of the 6 month period, samples from the dispersion were seeded on nutrient broth agar plates at the appropriate serial dilution. The number of colony forming units was determined for both samples. The measured concentration was 1 × 10 ⁷ in both cases. This shows the effectiveness of the formulation in preserving microorganisms. The above experiment was performed in comparison with Pseudomonas putida, but the same result was obtained.

  While the invention has been described with specific embodiments thereof, it will be appreciated that many alternatives and modifications and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives and modifications and variations that fall within the spirit and broad scope of the appended claims. All publications and patents and patent applications mentioned in this specification are specifically and individually indicated that each individual publication or patent or patent application is incorporated herein by reference. To the same extent as it was, it is incorporated herein by reference in its entirety. In addition, citation or identification of any reference in this specification shall not be construed as an admission that such reference is available as prior art to the present invention.

FIG. 1 is a schematic view of a dispenser according to the present invention.

Claims (35)

A probiotic composition comprising, as active ingredients, a viable Escherichia coli BU-230-98 strain (ATCC Deposit No. 202226 (DSM 12799)) and a formulation for maintaining the viability of the Escherichia coli strain. The formulation comprises at least one volatile fraction (VF) of the plant extract, the volatile fraction being under pressure below atmospheric pressure and at a bath temperature not exceeding 38 ° C. Obtained by steam distillation of the extract, at least one of the volatile fractions (VF) of the plant extract maintains the viability of the E. coli strain of 10 ⁷ cells / ml at room temperature for at least 2 months A probiotic composition, selected to allow.   The probiotic composition of claim 1, further comprising at least one flavoring agent.   The probiotic composition of claim 1, wherein the plant extract is obtained from a plant organ selected from the group consisting of leaves, stems, roots and fruits.   The probiotic composition of claim 1, wherein the plant extract is obtained from a plant selected from the group consisting of vegetables and herbs.   5. The probiotic composition of claim 4, wherein the vegetable is selected from the group consisting of soybean, alfalfa, garlic, beet and cabbage.   The probiotic composition of claim 4, wherein the herb is selected from the group consisting of parsley, mint and dill.   The probiotic composition of claim 1 further comprising a beehive product.   The probiotic composition of claim 7, wherein the bee nest product is propolis.   The probiotic composition of claim 1, wherein said composition prevents or treats a gastrointestinal infection or gastrointestinal abnormality.   2. The probiotic composition of claim 1, wherein the composition maintains or restores a normal gastrointestinal microflora.   2. The probiotic composition of claim 1, wherein the composition prevents or treats diarrhea.   2. The probiotic composition of claim 1, wherein the composition prevents or treats gastrointestinal infections caused by enteric pathogens.   13. The probiotic composition of claim 12, wherein the pathogen is a gram negative bacterium or a gram positive bacterium.   The probiotic composition of claim 1 comprising distilled water and a volatile fraction of beet, dill and parsley.   The probiotic composition of claim 1 comprising distilled water and a volatile fraction of beet, dill and grapefruit.   The probiotic composition of claim 1, wherein the composition prevents or treats a gastrointestinal Salmonella infection.   2. The probiotic composition of claim 1, wherein the composition prevents or treats infectious diarrhea, chronic diarrhea, or diarrhea resulting from antibiotic therapy or radiation therapy or chemotherapy.   The probiotic composition of claim 1, wherein the composition treats infectious diarrhea and the composition comprises distilled water and a volatile fraction of beet, dill and parsley.   The infectious diarrhea is C.I. difficile, Salmonella spp. 19. The probiotic composition of claim 18 caused by Shigella, Campylobacter, E. coli, Proteus, Pseudomonas, or Clostridium.   2. The probiotic composition of claim 1 that normalizes the physiological activity of the gastrointestinal tract.   A method for growing E. coli BU-230-98 strain (ATCC Deposit No. 202226 (DSM 12799)), wherein a starter culture of E. coli BU-230-98 strain (ATCC Deposit No. 202226 (DSM 12799)) is grown. Inoculating the medium and growing the E. coli strain until the optical density at 650 nm is 15-30. A method for producing a probiotic composition comprising viable E. coli strain BU-230-98 (ATCC Deposit No. 202226 (DSM 12799)), wherein the viability of said E. coli strain at 10 ⁷ cells / ml is Suspending in a formulation for maintenance at room temperature for at least 2 months, said formulation comprising at least one volatile fraction (VF) of the plant extract, said plant extract being water A process obtained by extraction, wherein the volatile fraction is prepared by steam distillation of the plant extract under a pressure below atmospheric pressure and at a bath temperature not exceeding 38 ° C.   23. The method of claim 22, wherein the plant extract is obtained from a plant organ selected from the group consisting of leaves, stems, roots and fruits.   23. The method of claim 22, wherein the plant extract is obtained from a plant selected from the group consisting of vegetables and herbs.   25. The method of claim 24, wherein the vegetable or herb is selected from the group consisting of soybean, parsley, mint, dill, alfalfa, garlic, beet and cabbage.   23. The method of claim 22, further comprising the addition of a beehive product.   27. The method of claim 26, wherein the beehive product is propolis.   23. The method of claim 22, wherein at least one of the volatile fractions is prepared from alfalfa, soybean, beet, dill and mint. A biological biocontrol method comprising the step of spraying E. coli BU-230-98 strain (ATCC Deposit No. 202226 (DSM12799)) in endemic areas, wherein the E. coli strain is 10 ⁷ cells / ml Present in a formulation containing at least one volatile fraction (VF) of a plant extract selected to maintain the viability of an E. coli strain at room temperature for at least 2 months, wherein the plant extract is A process obtained by water extraction, wherein the volatile fraction is prepared by steam distillation of the plant extract under a pressure below atmospheric pressure and at a bath temperature not exceeding 38 ° C. A method for removing oil contamination, comprising the step of spraying E. coli BU-230-98 strain (ATCC Deposit No. 202226 (DSM 12799)) in a contaminated area, wherein the E. coli strain is 10 ⁷ cells / ml Present in a formulation containing at least one volatile fraction (VF) of a plant extract that maintains the viability of the E. coli strain at room temperature for at least 2 months, said plant extract being obtained by water extraction Wherein the volatile fraction is prepared by steam distillation of the plant extract under a pressure below atmospheric pressure and at a bath temperature not exceeding 38 ° C. A method for reviving an organic gas decomposing microbial population in a biofilter, comprising a step of applying E. coli BU-230-98 strain (ATCC Deposit No. 202226 (DSM 12799)) to the filter, wherein the E. coli strain comprises 10 ⁷ cells. Present in a formulation containing at least one volatile fraction (VF) of a plant extract that maintains viability of the E. coli strain at room temperature for at least 2 months, wherein the plant extract is water A process obtained by extraction, wherein the volatile fraction is prepared by steam distillation of the plant extract under a pressure below atmospheric pressure and at a bath temperature not exceeding 38 ° C.   A food product supplemented with viable E. coli strain BU-230-98 (ATCC Deposit No. 202226 (DSM 12799)). 33. The food product of claim 32 comprising at least one substance obtained from milk. 34. The food product of claim 33 , selected from the group consisting of cheese and yogurt.   A method for preparing a starter product for a fermentation process, wherein an amount of E. coli BU-230-98 (ATCC Deposit No. 202226 (DSM 12799)) sufficient to induce a fermentation process is grown and said E. coli Suspending a strain in a formulation containing at least one volatile fraction (VF) of a plant extract that maintains the viability of the E. coli strain for at least 2 months at room temperature, A process wherein the extract is obtained by water extraction and the volatile fraction is prepared by steam distillation of the plant extract under a pressure below atmospheric pressure and at a bath temperature not exceeding 38 ° C.

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