NZ544366A - Bacterial strain, processed plant extracts, compositions containing same, processes for their preparation and their therapeutic and industrial applications - Google Patents

Abstract

The use of a formulation comprising at least one volatile fraction (VF) of a plant extract, said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 degrees C; to sustain viability of as much as 107 cells/ml of at least one microorganism for at least two months at room temperature, with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799). Also disclosed are dispensers for said formulation and methods of using said formulation.

Description

*10050224392* Patents Form No. 5 5443 66 N.Z. No, Divided out of Parent Application No. 526748 NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION BACTERIAL STRAIN, PROCESSED PLANT EXTRACTS, COMPOSITIONS CONTAINING SAME, PROCESSES FOR THEIR PREPARATION AND THEIR THERAPEUTIC AND INDUSTRIAL APPLICATIONS We, The Bio Balance Corp., an American company of 16 East 34th Street, New York, NY 10016, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - (Followedby 1A) i,>■':*i ov • V 21 2035 1A BACTERIAL STRAIN, PROCESSED PLANT EXTRACTS, COMPOSITIONS CONTAINING SAME, PROCESSES FOR THEIR PREPARATION AND THEIR THERAPEUTIC AND INDUSTRIAL APPLICATIONS 5 The present application is a divisional of NZ 526748.

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to (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 10 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 15 the formulation of the invention and microorganism(s) of industrial applicability; and (vi) industrial processes and apparatuses in which the latter composition is used.

Under normal conditions, the gastrointestinal (GI) tract microflora contributes significantly to the health and well being of an individual. It is well 20 known that the microflora is a complex and diverse population of microorganisms, which may have both beneficial and harmful effects on the individual. Under normal gestation conditions, the fetus in utero is sterile, but on passage through the vagina during birth it acquires microorganisms resulting in the formation of a gut microflora. The final indigenous gut microflora which stabilizes in the gut is a very 25 complex collection of over one thousand "different types of microorganisms, consisting of about 400 different types of bacteria [Fuller R. J. Applied Bacteriology 66:365-378 (1989)]. The composition of the gut microflora is determined by both host and microbial factors, and although there are a lot of bacteria which can survive and grow in the GI tract, there are many which cannot. 30 In addition, the surviving organisms have to avoid the effect of peristalsis which normally flushes out the bacteria with the food. This may be achieved by the bacteria immobilizing themselves by attachment to the gut wall, and/or by multiplying at a rate that exceeds the rate of removal by peristalsis.

In general, the presence of gut microflora is symbiotic as the microflora not 35 only assists in disintegrating some undigestable foods, it also protects the individual from infections caused by pathogens. This latter phenomenon has been described under such names as 'bacterial antagonism', 'bacterial interference', 'barrier effect', 'colonization resistance', 'competitive exclusion' and many others. •afcu±w»Au PF<0PEfm QFHCI OF N.Z. 11 APR 2007 r.fiivec 2 The gut protective microflora is very stable. However, it is less effective in the young, elderly and the compromised patient. Further, it can be influenced by certain dietary and environmental factors, the three most important being excessive hygiene, antibiotic therapy and stress.

Under conditions where the balance of the gut microflora is adversely affected, probiotics become of potential value in restoring the gut microflora and enabling the individual host to return to normal.

Probiotics are a class of microorganisms defined as live microbial organisms that beneficially affect the animal and human hosts. The beneficial effects include 10 improvement of the microbial balance of the intestinal microflora or improving the properties of the indigenous microflora. The beneficial effects of probiotics may be mediated by a direct antagonistic effect against specific groups of organisms, resulting in a decrease in numbers, by an effect on their metabolism or by stimulation of immunity. Probiotics may suppress viable counts of an undesired 15 organism by producing antibacterial compounds, by competing for nutrients or for adhesion sites. Further, they may alter microbial metabolism by increasing or decreasing enzyme activity or they may stimulate the immune system by increasing antibody levels or increasing macrophage activity.

W095/16461 describes- a probiotic composition of anaerobic bacteria 20 effective in controlling or inhibiting Salmonella colonization in domesticated animals. The probiotic composition includes populations or cultures of 29 substantially biologically pure bacteria, inter alia, E. coli. However, the suppression of the pathogen by the probiotic composition described in this PCT publication requires the combined action of a large number of bacterial strains. 25 W097/35596 describes the administration of a freshly prepared probiotic mixture obtained by mixing a powder containing Lactobacillus reuteri, Lactobacillus acidophilus and Bifidobacterium infantis with a liquid. The mixture is described to be effective in preventing infectious diarrhea or diarrhea caused by antibiotic therapy in humans. The freeze-dried live bacteria are, however, in 30 anabiotic state. The need to wet the microorganism before administration, in order to reinstate its vitality, is a disadvantage, since normally many bacteria do not survive the re-hydration. Moreover, the surviving organisms are not immediately metabolically active, and cannot survive the extreme, acidic conditions of the stomach. Furthermore, when administered to a recipient with diarrhea, the rate of 35 their removal from the gut may exceed the rate of reinstation of viability, resulting in minimal or no beneficiary effect. i IHTpiIFCTUAl PHOPEfflV 0FFICE| "N 0FN2. 1 17 APR 2007 received 3 Preservation of viability and conservation of the activity of probiotic organisms by their formulation is the issue of numerous publications. W098/26787 describes the enhancement of a resident population of lactic acid-producing microorganisms, preferably lactobaccillii, in the GI tract of an animal by providing 5 the same with p-glucan, optionally in combination with prebiotic and/or probiotic microorganisms.

W097/34591 also describes the enhancement of resident population of microorganisms, or the suppression of the undesired resident population at a selected site of the GI tract of an individual, by providing the individual with a 10 selected modified or unmodified starch or mixtures thereof, which act as carrier for one or more probiotic microorganisms and as a growth or maintenance medium for the microorganisms. The probiotic elements are bound to the carrier in a manner so as to protect the microorganisms during passage to the large bowel or other regions of the GI tract.

Microorganisms are used in a variety of industrial processes. These processes can be divided into continuous processes and batch processes. In continuous processes, such as a continuous aerobic or anaerobic fermentation line, used for the production of a variety of fermentation products, including, for example, ethanol and methanol, a fermentable raw material is continuously fed into 20 the line and the fermentive product is continuously collected from the line, in a controlled fashion, such that the microorganism population in the line is sustained throughout the process. In batch processes, such as, but not limited to, batch aerobic or anaerobic fermentation, biodegradation of oil in, for example, oil spills, and the like, a fermentable raw material is mixed with a population of 25 microorganism(s) so as to instate fermentation. Both in continuous fermentation and in batch fermentation processes, it is desired, in some cases, to start or reinstate the process with a large and viable population of the relevant microorganism(s). For example, when biodegrading oil, it is advantageous to start the process with an as large as possible population of the microorganisms Pseudomonas spp. or 30 Alcaligenes spp., for example, so as to minimize the time for complete biodegradation of the oil. When using filters enriched with microorganism(s) for biodegradation of organic fumes or volatiles, the result is a continuous process in which polluting compounds are degraded into harmless compounds. However, the microorganisms present in the filter depend for their survival on continuous supply 35 of organic fumes or volatiles. Nevertheless, in some instances the supply of organic fumes or volatiles is discontinued and, as a result, the microorganism(s) are lost. Under such circumstances, reinstation of an effective viable amount of INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 APR 2007 RECEIVED 4 microorganism(s) in the filter is required in a short period of time, so as permit immediate restoration of a functional filter.

There is thus a widely recognized need for, and it would be highly advantageous to have, a formulation in which microorganisms, including probiotic 5 microorganisms and microorganisms useful in a variety of industrial applications, could be maintained viable and in a metabolically active form for long periods of time.

While reducing the present invention to practice, it has been unexpectedly found that a single species of a non-pathogenic probiotic microorganism derived 10 from E. coli is, alone, capable of restoring normal GI flora of man and of a variety of mammals and avians. It has also been surprisingly found that this microorganism, as well as other microorganisms, could be preserved for long periods of time, in a viable and metabolically active form, in a formulation comprising water solution of volatile fraction(s) of various plant extracts. A 15 probiotic composition comprising the probiotic organism suspended in the formulation was found to be effective in the treatment and prevention of various gastrointestinal disorders. It has further been unexpectedly found that the formulation per se is effective as a body weight gain enhancer and as an immuno-stimulator in mammals and avians.

SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided an Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799). 25 According to another aspect of the present invention there is provided a probiotic composition comprising viable Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799) and a formulation for maintaining viability of the Escherichia coli strain. The probiotic composition preferably further comprising at least one flavouring agent. Preferably, the formulation includes at 30 least one volatile fraction (VF) of a plant extract prepared by steam distillation of a plant (e.g., beet, dill, parsley or grapefruit) extract under reduced pressure and at a bath temperature preferably not exceeding 38 °C.

The probiotic composition of the present invention may be identified for preventing or treating gastro-enteric infections or disorders, maintaining or 35 reinstating normal gastro-intestinal microflora, preventing or treating diarrhea, preventing or treating gastro-enteric infection caused by an enteric pathogen, such as a Gram negative bacterium or Gram positive bacterium, preventing or treating INTELLECTUAL PROPERTY OFFlUt OF N.Z. 17 APR 2007 RECEIVED gastro-enteric Salmonella infection, preventing or treating infectious diarrhea, caused by, for example C. difficile, Salmonella, particularly S. Shigella, Campylobacter, E. coli, Proteus, Pseudomonas or Clostridium, chronic diarrhea or diarrhea resulting from antibiotic therapy, radiotherapy or chemotherapy, and/or for 5 normalizing the physiological activity of the gastrointestinal tract.

According to yet another aspect of the present invention there is provided a formulation comprising at least one volatile fraction (VF) of a plant extract, the volatile fraction is prepared by steam distillation of the plant extract under reduced pressure and at a bath temperature not exceeding 38 °C. The plant extract is 10 preferably obtainable from a plant organ selected from the group consisting of leaves, stems, roots and fruit. The plant can be a vegetable, such as soy bean, alfalfa, garlic, beet and cabbage, or a herb, such as parsley, mint and dill. The formulation may further include a beehive product, such as, but not limited to, propolis. The formulation can serve as a veterinary feed or food additive for 15 enhancing animal weight gain in a recipient. It can also serve to preserve viable 1 bacteria as is further described herein.

Hence, according to an additional aspect of the present invention there is provided a process of preparing a volatile fraction of a plant, comprising the steps of (a) grinding a plant derived material to give a plant biomass; (b) mixing the plant 20 biomass with water and stirring at ambient temperature; (c) steam distilling the mixture obtained in step (b) under reduced pressure and at a bath temperature not exceeding 38°C; and (d) collecting volatile fraction obtained from the steam distillation. Preferably, the reduced pressure is of 5-10 mbar. This formulation can be used as a food additive, a feed additive and has unexpected therapeutic uses, as 25 well as advantageous bacteria preservation properties. A therapeutic formulation containing the volatile fraction of a plant is also disclosed and may be prepared by mixing at least one volatile fraction (VF) of a plant extract with physiologically or veterinary acceptable additives, carriers or diluents. Preferably, the volatile fraction is prepared by steam distillation of the plant extract under reduced pressure and at a 30 bath temperature not exceeding 38 °C.

Thus, according to still another aspect of the present invention there is provided a probiotic composition comprising (a) an effective amount of at least one viable probiotic microorganism having a beneficial biological or therapeutic activity in the gastrointestinal tract; and (b) at least one volatile fraction (VF) of a 35 plant extract preferably prepared as described herein. The probiotic microorganism can be E. coli, preferably, strain BU-230-98, ATCC Deposit No. 202226 and it can be identified for treatment or prevention of anv of the above riiiScTUM^opiS^^ OF N.Z 17 APR w RECEIVED- 6 disorders/pathologies/diseases/syndromes and in addition or as an alternative in can be identified for treatment or prevention of dyspeptic symptoms, for enhancing the immune response in a patient suffering from an immune disorder, resulting from, for example, immune-response suppression therapy.

According to still another aspect of the present invention there is provided a composition comprising (a) at least one microorganism, such as, Escherichia spp., Alcaligenes spp., Arthrobacter spp., Bifidobacterium spp., Lactobacillus spp., Lactococcus spp., Nitrosomonas spp. and Pseudomonas spp.; and (b) at least one volatile fraction (VF) of a plant extract, the at least one volatile fraction is selected 10 so as to sustain viability of the at least one microorganism for at least 2 months, preferably at least 3-6 months, more preferably 6-12 months or more at room temperature.

According to a particular aspect of the present invention, there is provided a probiotic composition comprising (a) at least one microorganism, with the proviso 15 that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799); and (b) at least one volatile fraction (VF) of a plant extract, said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, wherein 20 said at least one volatile fraction is selected so as to sustain viability of as much as 107 cells/ml of said at least one microorganism for at least two months at room temperature.

According to another aspect of the present invention there is provided a process of growing Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 25 (DSM 12799), the process comprising the step of seeding a starter cloture of Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799) into a growth media and growing the Escherichia coli strain up to optical density of 15-30 at 650 nm.

According to yet another aspect of the present invention there is provided a 30 process of manufacturing a probiotic composition, the process comprising the step of suspending viable Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799) in a formulation for maintaining viability of the Escherichia coli strain. Preferably, the formulation includes at least one volatile fraction (VF) of a plant extract, prepared, preferably by steam distillation of the plant extract under 35 reduced pressure and at a bath temperature not exceeding 38 °C, obtained from, for example, a plant organ selected from the group consisting of leaves, stems, roots INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 APR 2007 received 7 and fruit, either a vegetable and a herb, such as soy bean, parsley, mint, dill, alfalfa, garlic, beet or cabbage.

According to still another aspect of the present invention there is provided a process of preparing a probiotic composition, the process comprising the step of suspending an effective amount of at least one viable probiotic microorganism having a beneficial biological or therapeutic activity in the gastrointestinal tract in an formulation containing at least one volatile fraction (VF) of a plant extract.

According to an additional aspect of the present invention there is provided a process of preparing a composition including viable microorganisms, the process comprising the step of suspending at least one microorganism in a formulation containing at least one volatile fraction (VF) of a plant extract, the at least one volatile fraction is selected so as to sustain viability of the at least one microorganism for at least 2 months at room temperature. The microorganism can be, for example, Escherichia spp., Alcaligenes spp., Arthrobacter spp., Bifidobacterium spp., Lactobacillus spp., Lactococcus spp., Nitrosomonas spp. or Pseudomonas spp.

According to a particular aspect of the present invention, there is provided a process of preparing a probiotic composition including microorganisms, the process comprising suspending at least one microorganism in a formulation containing at least one volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction sustains viability of as much as 107 cells/ml of said at least one microorganism for at least two months at room temperature, with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).

According to yet an additional aspect of the present invention there is provided a dispenser of microorganisms comprising a reservoir and a dispensing mechanism being connected thereto, the reservoir housing at least one microorganism in a formulation containing at least one volatile fraction (VF) of a plant extract, the at least one volatile fraction is selected so as to sustain viability of the at least one microorganism for at least 2 months at room temperature.

According to a particular aspect of the present invention, there is provided a dispenser of microorganisms comprising a reservoir and a dispensing mechanism being connected thereto, said reservoir housing at least one microorganism, in a formulation containing at least one volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water extra action being 8 prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction is in an effective amount to sustain viability of said at least one microorganism for at least two months at room temperature, with the proviso that 5 said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).

According to still an additional aspect of the present invention there is provided a method of biocontrol of an organism, the method comprising the step of spreading in an endemic area at least one viable microorganism capable of 10 biocontrolling the organism, the at least one viable microorganism being in a formulation containing at least one volatile fraction (VF) of a plant extract, the at least one volatile fraction is selected so as to sustain viability of the at least one microorganism for at least 2 months at room temperature.

According to a particular aspect of the present invention, there is 15 provided a method of biocontrol of an organism, the method comprising the step of spreading in an endemic area at least one viable microorganism capable of biocontrolling the organism, said at least one viable microorganism being in a formulation containing at least one volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water extraction and said volatile fraction being 20 prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction is selected so as to sustain viability of as much as 107 cells/ml of said at least one microorganism for at least two months at room temperature, with the proviso that said microorganism is not Escherichia coli strain BU-230-98 25 ATCC Deposit No. 202226 (DSM 12799).

According to a further aspect of the present invention there is provided a method of eliminating an oil pollution, the method comprising the step of spreading in a polluted area at least one viable microorganism capable of degrading oil, the at least one viable microorganism being in a formulation 30 containing at least one volatile fraction (VF) of a plant extract, the at least one volatile fraction is selected so as to sustain viability of the at least one microorganism for at least 2 months at room temperature.

According to a particular aspect of the present invention, there is provided a method of eliminating an oil pollution, the method comprising the step of spreading 35 in a polluted area at least one microorganism capable of degrading oil, said at least one viable microorganism being in a formulation containing at least one volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water jUTfi IPCTUAL PROPERTY umut OF N.Z. 17 APR 2007 received 9 extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction is selected so as to sustain viability of as much as 10 cells/ml of said at least one microorganism for 5 at least two months at room temperature, with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).

According to yet a further aspect of the present invention there is provided a method of restoring organic fumes degrading microorganism population in a biofilter, the method comprising the step of dispensing onto the filter at least one 10 viable microorganism capable of degrading organic fumes, the at least one viable microorganism being in a formulation containing at least one volatile fraction (VF) of a plant extract, the at least one volatile fraction is selected so as to sustain viability of the at least one microorganism for at least 2 months at room temperature.

According to a particular aspect of the present invention, there is provided a method of restoring organic fumes degrading microorganism population in a biofilter, the method comprising dispensing onto the filter at least one microorganism capable of degrading organic fumes into environmentally harmless compounds, said at least one microorganism being in a formulation containing at 20 least one volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction is selected so as to sustain viability of as much as 107 cells/ml of said at least one 25 microorganism for at least two months at room temperature, with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).

According to still a further aspect of the present invention there is provided a method of preparing a starter for a fermentation process, the method comprising the 30 step of growing a sufficient amount of a starter microorganism and suspending the starter microorganism in a formulation containing at least one volatile fraction (VF) of a plant extract, the at least one volatile fraction is selected so as to sustain viability of the starter microorganism for at least 2 months at room temperature.

According to a particular aspect of the invention, there is provided a method 35 of preparing a starter for a fermentation process, the method comprising growing an amount of a starter microorganism sufficient to induce a fermentation process, and suspending said starter microorganism in a formulation containing at least one 1 OF N1 \ 7 APR 2007 RECEIVES. volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction is selected so as 5 to sustain viability of as much as 107 cells/ml of said at least one microorganism for at least two months at room temperature, with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).

BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings: FIG. 1 is a schematic depiction of a dispenser according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION ^ 25 The present invention is of (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-30 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. 35 The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions. 1NTEI1FP-TUAL PROPER OFFICfc OF N.Z. 17 APR 2007 RECEIVED 11 Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

In one aspect, the present invention relates to a probiotic microorganism being a non-pathogenic bacterium derived from E. coli, having a beneficial physiological and/or therapeutic activity in, for example, the GI tract and deposited 10 at the ATCC under deposit No. 202226 and at DSMZ under deposit No. 12799.

"Probiotic" is used herein as an adjective to describe an isolated bacteria having the property of inhibiting the growth of at least one pathogen. The test of an inhibition used herein was an in vitro test on solid medium in which culture supernatants of candidate isolated bacteria were observed for their property of 15 inhibiting the growth of a pathogen when applied to the surface of the solid medium. 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. Probiotic bacterial supernatants caused a ring of clear agar or of reduced growth density indicating inhibition of the pathogen in the vicinity of the disc. 20 There are other tests for inhibition which are available or could be devised, including direct growth competition tests, in vitro or in vivo which can generate a panel of probiotic bacteria similar to that described herein. The bacterial strains identified by any such test are within the category of probiotic bacteria, as the term is used herein.

The probiotic bacteria of the present invention may serve as a food or feed additive, so as to provide food or feed product supplemented with viable Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799). A food product of the present invention may further include at least one milk derived substance and may be selected from the group consisting of a cheese and a yogurt. 30 The present invention further relates to a formulation comprising at least one volatile fraction (VF) of a plant extract, the volatile fraction being prepared by distillation of the plant extract under reduced pressure and at a bath temperature typically not exceeding 38 °C.

The plant matter from which the volatile fraction may be obtained may be 35 any suitable plant part such as fruit, leaf, stem or root. Many plants are suitable as a source for the volatile fractions, for example apple, citrus, soy bean, beet, cabbage, garlic and alfalfa, as well as herbs such as parsley, mint and dill. The formulations INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 APR 2007 received 12 of the invention may optionally further comprise a suitable amount of a volatile fraction of an apicultural product such as honey, propolis or other beehive product, which may be prepared in the same manner as described herein for obtaining volatile fractions from plant extracts.

As will be described in more detail in the following Examples, the formulation itself may be used as a food/feed additive. It has been found by the inventors to have a weight gain enhancing activity and it constitutes an aspect of the invention. According to a particular embodiment of this aspect of the invention, the animal weight gain enhancing formulation or feed additive comprises distilled 10 water and volatile fractions of alfalfa, soy beans, beet and dill^ preferably at a volume ratio of the volatile fractions of 2:8:1:4. Alternatively, a formulation or feed additive of the present invention comprises distilled water and volatile fractions of carrot, beet, dill and grapefruit (1 part each) per 100 parts of water. Still alternatively, a formulation or food additive of the present invention comprises 15 distilled water and volatile fractions of beet, dill and parsley (1 part each) per 100 parts of water.

In a further aspect, the invention relates to a composition comprising the above formulation of the invention and at least one viable microorganism, either a probiotic organism having a therapeutic or beneficial biological activity in, for 20 example, the GI tract of human and/or animal, to thereby provide a probiotic composition, or an organism having industrial applications, to thereby provide a composition of industrial applicability.

A particular advantage of the probiotic and industrial compositions of the invention stems from the fact that they are liquid preparations. Being under 25 biologically active conditions, the formulation serves also as a supportive medium for living bacteria, as opposed to lyophilized formulations where the bacteria are in an anabiotic condition. As a result, the probiotic composition of the invention, for example, is active immediately following oral administration, beginning with the upper portion of the GI tract, where primary effects of the majority of intestinal 30 pathogens take place, causing development of adverse gastro-enteric syndromes. The probiotic compositions of the invention may also be used as body weight-increasing preparations or food/feed additives. Similarly, the industrial composition of the invention can be used to efficiently reinstate a population of microorganisms of industrial applicability.

Thus, it is noted that, for probiotic applications, probiotic bacteria other than such belonging to the strain BU-230-98 (ATCC Deposit No. 202226, also deposited at the DSM under No. 12799), may be used with the plant material derived Intellectual proper™ of n.z. i 17 APR 2M7 qpcfived 13 formulation of the invention. Such probiotic bacteria have a very broad spectrum of antagonistic activity. They also belong to the same phylogenetic group of the majority of intestinal pathogens and share the same systems of survival. Therefore, the suppression and exclusion of intestinal pathogens may include many different 5 mechanisms, for example, secretion of antagonistic material, competition for utilization of nutrients and competition for adhesion receptors. Thus, any nonpathogenic bacteria which comply with these criteria, may be used in the probiotic compositions of the invention.

According to a further aspect of the invention, the probiotic compositions of 10 the invention may be used for preventing or treating gastro-enteric infections. Term 'gastro-enteric infection' is to be taken to mean any infection caused an enteric pathogen, including, inter alia, Gram negative and Gram positive bacteria. By improving the general balance and health of the GI tract, the formulations and probiotic compositions of the invention may be instrumental in prophylaxis of also 15 GI infections caused by yeast, viruses and protozoa.

The term 'therapeutically effective amount' or 'effective amount' for purposes herein is the amount determined by such considerations as are known in the art. The amount must be sufficient to enable the efficient restoration of the GI microflora thus leading to the normalization of the function of the GI tract. 20 In a particular embodiment, the probiotic composition for preventing the development of gastro-enteric infections comprises the formulation of the invention made from distilled water and volatile fractions of alfalfa, soy beans, beet, dill and mint as defined herein, at a volume ratio of, e.g., 5:1:5:15:1, and a therapeutically effective amount of a probiotic bacteria such as E. coli ATCC Deposit No. 202226 25 (identical with DSM 12799) or any other probiotic bacteria.

A specific example for an gastro-enteric infection is that caused by Salmonella and the invention is of particular advantage in preventing or treating gastro-enteric infections caused thereby.

Further, the food additive or formulation of the invention, alone or in 30 combination with an effective amount of a probiotic microorganism, such as the E. coli strain ATCC Deposit No. 202226 (identical with DSM 12799), may be used for treating or preventing infectious diarrhea, chronic diarrhea or diarrhea caused by antibiotic or chemo-therapy.

According to a further specific embodiment of the invention, such a 35 probiotic composition for treating diarrhea may comprise distilled water and volatile fractions of alfalfa, soy beans, beet, dill and mint at a volume ratio of, e.g., INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 APR 2<W received 14 :1:5:15:1, and a therapeutically effective amount of the ATCC No. 202226 (DSMZ 12799).

The infectious diarrhea may be caused by numerous factors, for example, by a microorganism selected from C. difficile, Salmonella, particularly S. Shigella, 5 Campylobacter, E. coli, Proteus, Pseudomonas, Clostridium, enteric Staphylococcus. These are but few of many infecting agents.

Yet further, the probiotic compositions of the invention may be used for effectively restoring the GI microflora in a subject in need of such treatment which leads to the normalization of the function of the GI tract. Such compositions may 10 include, for example, distilled water and volatile fractions of alfalfa, soy bean, beet, dill, mint, parsley and cabbage, preferably at a ratio of volatile fractions of, e.g., 1:5:5:2:2:1, and a therapeutically effective amount of the probiotic bacteria ATCC Deposit No. 202226 (DSM 12799).

Other purposes for which the probiotic compositions of the invention, 15 comprising at least one probiotic bacteria having a therapeutic effect in, for example, the GI tract, include alleviation of lactose intolerance in subjects suffering from lactose intolerance, treatment of enterocolitis, treatment of constipation, for reduction of cholesterol levels in the blood, for treatment of dyspeptic symptoms, and/or for stimulation of the immune system in subjects suffering from an immune 20 system disorder, which may be an immune disorder caused by immuno-suppressive therapy.

In a different aspect, the invention relates to a process for preparing a volatile fraction of a plant extract, which process comprises the steps of: (a) grinding plant derived matter to obtain a plant biomass; (b) mixing the plant 25 biomass obtained in step (a) with water at a weight proportion of preferably 3 parts water to 1 part of the plant biomass and stirring the same for at least 2 hours at ambient temperature; (c) distilling the mixture obtained in step (b) under reduced pressure and at a bath temperature preferably not exceeding 38 °C; and (d) collecting the volatile fraction obtained from the steam distillation, which 30 fraction may further be diluted in a suitable buffer.

The volatile fraction of the invention may be mixed with water to give the food/feed additive or formulation of the invention. The food/feed additive or formulation of the invention may also be prepared by mixing more than one plant volatile fraction obtained by the process of the invention. This mixture may be 35 further mixed with water.

The volatile fractions may be prepared from may any suitable fruit, vegetable, leaf, stem or root of a plant. The plant can be, for example, apple, citrus INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 APR 2007 received fruit, soy bean, beet, garlic, cabbage or alfalfa, or a herb such as parsley, mint or dill. It should be noted that when appropriate, the formulation may further comprise volatile fractions from apicultural products such as honey or propolis or other beehive products. These volatile fractions may be prepared in the same 5 manner of the plant extract volatile fractions.

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

As indicated hereinbefore, the food/feed additive or formulation of the invention may be further combined with at least one probiotic or industrial agent, to 10 give the probiotic or industrial compositions of the invention. Thus, the process of preparation of the invention may also further comprise the step of suspending at least one viable probiotic or industrial microorganism in the volatile fraction obtained in said step (d) or in a mixture of such volatile fractions obtained as described above.

According to a particular process of the invention, the probiotic microorganism suspended may be the novel bacterium of the invention, derived from E. coli and deposited at the ATCC under Deposit No. 202226 (and at DSM under deposit No. 12799).

Finally, the invention relates to the use of a volatile fraction (VF) of a plant 20 extract in the preparation of a food/feed additive, wherein the VF is prepared by steam distillation of said plant extract or from apiculture product extract such as honey or propolis at a bath temperature not exceeding 38 °C.

An additional aspects of the invention include a process of preparing a composition including viable microorganisms. The process is effected by 25 suspending at least one microorganism in a formulation containing at least one volatile fraction (VF) of a plant extract. The formulation is selected so as to sustain viability of the microorganism(s) for at least 2 months at room temperature. The microorganism of choice may be probiotic or have industrial applicability. Organisms which can be maintained in a formulation of the present invention 30 include Escherichia spp., Alcaligenes spp., Arthrobacter spp., Bifidobacterium spp., Lactobacillus spp., Lactococcus spp., Nitrosomonas spp. and Pseudomonas spp.

As shown in Figure 1, in another aspect the present invention provides a dispenser 10 of microorganisms. Dispenser 10 includes a reservoir 12 and a dispensing mechanism 14 connected thereto. Reservoir 12 houses at least one 35 microorganism in a formulation containing at least one volatile fraction (VF) of a plant extract. The formulation is selected so as to sustain viability of the microorganism(s) for at least 2 months at room temperature. Reservoir 12 may be INTELLECTUAL PROPtHU uI-HULI OF N.Z. 17 APR W rfT.EIVED 16 refrigerated, say to about 2-20 °C, preferably to about 4 °C. Dispenser 10 may be an airborne dispenser, so as to allow dispersion of pest control bioagents. The dispenser may be hand held. In any case, the dispenser may be designed to automatically dispense a predefined volume therefrom, e.g., periodically. To this 5 end, dispenser 10 may include a programmable or preset control mechanism 16, valves 18, etc.

Thus, according to another aspect, the present invention provides a method of biocontrol of an organism which is effected by spreading in an endemic area at least one viable microorganism capable of biocontrolling the organism. The viable 10 microorganism(s) are present in a formulation containing at least one volatile fraction (VF) of a plant extract selected so as to sustain viability of said at least one microorganism(s) for at least 2 months at room temperature.

Still according to another aspect the present invention provides a method of eliminating an oil pollution which is effected by spreading in a polluted area at least 15 one viable microorganism capable of degrading oil, such as Pseudomonas spp. The viable microorganism(s) is present in a formulation containing at least one volatile fraction (VF) of a plant extract selected so as to sustain viability of said at least one microorganism for at least 2 months at room temperature.

Yet according to another aspect the present invention provides a method of 20 restoring organic fumes degrading microorganism population in a biofilter by dispensing onto the filter at least one viable microorganism, such as Pseudomonas spp., Nitrosomonas spp., or Arthrobacter spp. capable of degrading organic fumes. The viable microorganism(s) is present in a formulation containing at least one volatile fraction (VF) of a plant extract selected so as to sustain viability of said at 25 least one microorganism for at least 2 months at room temperature.

Still according to another aspect of the present invention there is provided a method of preparing a starter for a fermentation process by growing a sufficient amount of a starter microorganism, such as, Lactococcus spp., Escherichia spp., Bifidobacterium spp., Lactobacillus or Pseudomonas spp. and suspending the 30 starter microorganism in a formulation containing at least one volatile fraction (VF) of a plant extract selected so as to sustain viability of said starter microorganism for at least 2 months at room temperature.

Additional objects, advantages, and novel features of the present invention 35 will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated wtellectual^ropertv office of n.i 17 APR 2007 received 17 hereinabove and as claimed in the claims section below finds experimental support in the following examples.

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

Example 1 Preparation of a "volatile fraction " ofplant extracts 10 or extracts from apiculture products Fresh vegetables obtained from commercial markets were thoroughly washed by tap water, chopped and finely ground in an industrial blender. Distilled ' water was added to the vegetable biomass at the proportion of 3 parts water to 1 part of the vegetable mass by weight and left under stirring for at least 2 hours at 15 ambient temperature.

The mixture was then transferred into the evaporating flask of a rotatory evaporator and was evaporated at a reduced pressure (5-10 mbar) under such conditions that the temperature of the water bath did not exceed 38 °C, and that of the condenser inlet was 2-5 °C. About 1 liter of the condensate (volatile fraction) 20 were collected per 2.5 kg of plant biomass. This material could be preserved under refrigeration for at least 12 months without loosing its properties, as is further exemplified hereinunder.

The same procedure may be carried out, replacing the plant material with apiculture products such as honey or propolis.

Usually, each "volatile fraction" was prepared from a single kind of vegetable and used in different dilution to prepare different mixtures for various purposes.

INTELLECTUAL PROPERTY OFFICE OF N.Z 17 APR Ml received 18 Example 2 Isolation and growth of the probiotic organism E. coli A TCC Deposit No. 202226 The probiotic organism E. coli (deposited at the ATCC under No. 202226 on 5 May 3, 1999 and at the DSM under No. 12799 on May 4, 1999) was isolated from E. coli M-17 by sequential transfer of isolates initiating from long term (2 months) survivors preserved in a formulation containing volatile fractions of plant derived material as is described under Example 1 above at 37 °C and selecting for isolates capable of competition with pathogens as is further detailed below. The probiotic 10 organism E. coli (deposited at the ATCC under No. 202226 on May 3, 1999 and at the DSM under No. 12799 on May 4, 1999) was grown in the following medium: (NH4)2S04 - 5 g/liter, KH2PO4 - 13 g/liter, Na2HP04 - 13 g/liter, MgCl2 - 3 g/liter, CaCl2 - 0.3 g/liter, yeast extract - 10 g/liter, Soy peptone - 10 g/liter and glucose - 5 g/liter. Additional nutrients (yeast extract - 1 g/liter, Soy peptone - 2.5 15 g/liter and glucose - 90 g/liter) were continuously added following the growth of the culture in such a way that the glucose concentration in the fermentation broth was kept at a level of 2 g/liter. The pH of the fermentation broth was kept neutral by the continuous addition of 4N NH4OH. Culturing was carried out at 30 °C in a standard fermentation vessel with aeration of 0.5 wm for 16 hours, when the 20 growth became confluent. This procedure resulted in 10^-10^ cells/ml. The E. coli cells were harvested by centrifugation, resuspended in saline and re-precipitated. The microbial biomass could be kept in saline in the refrigerator for 48 hours without loosing viability.

Example 3 Preparation offood/feed additive formulation for the accelerated increase of body weight in mammals and avians The mixture contained volatile fractions of: alfalfa - 50 ml/liter, soy beans -200 ml/liter, beet - 25 ml/liter and dill - 100 ml/liter, prepared as described under 30 Example 1 above. The balance was made up by the distilled water.

Example 4 Preparation of a food supplement for the normalization of the function of the human GI tract 35 The mixture contained volatile fractions of: alfalfa - 50 ml/liter, soy beans - ml/liter, beet - 50 ml/liter, dill - 50 ml/liter, mint - 20 ml/liter, parsley - 20 ml/liter and cabbage - 10 ml/liter, prepared as described under Example 1 above.

Intellectual property office of n.z. 17 APR 2007 received 19 The balance was made up by distilled water. E. coli (ATCC 202226) cells 7 (Example 2) were suspended in the mixture at a cell concentration of 10 cells/ml. NaCl (e.g., 4-10 g/liter) may be optionally added for the improvement of taste.

Example 5 Preparation of a feed additive formulation for the prophylaxis of GI infections in mammals and avians The mixture contained volatile fractions of: alfalfa - 50 - ml/liter, soy beans -10 ml/liter, beet - 50 ml/liter, dill - 150 ml/liter and mint - 10 ml/liter, prepared as 10 described under Example 1 above. The balance was made by distilled water. E. coli (ATCC 202226) cells (Example 2) were suspended in the mixture at a cell 7 concentration of 10 cells improvement of the taste. 7 concentration of 10 cells/ml. NaCl (4-10 g/liter) may be optionally added for the Example 6 Antagonistic activity of E. coli (ATCC202226) against Salmonella typhymirium (ATCC 14028) Petri plates containing Modified Brilliant Green Agar, a selective growth medium for Salmonella, were inoculated with S. typhimirium. A 9 mm diameter 20 well was made in the agar. A volume of the food supplement (Example 4) was deposited in each well, and the plates were incubated for 24 hours at 35 °C. The same was repeated, but instead of the food supplement, the fluid obtained by its filtration through a microbiological filter membrane (pore size of 0.45 ]iim) was deposited in the well.

Around each well containing the feed additive an inhibition zone (10-17 mm) devoid of S. typhimirium colonies was observed. No inhibition zone was observed around the wells containing the filtrate free of the probiotic organism.

Example 7 Antagonistic activity of E. coli ATCC 202226 as well as of its parent strain E. coli M-17 against Shigella sps.

Cultures of S. flexneri, S. sonnei, E. coli (ATCC 202226) and M-17 were grown separately on a Nutrient Agar for 18-20 hours at 37 °C. All the cultures were harvested in saline and diluted to an optical density of 10 Klett units. Aliquots 35 of the diluted cultures of Shigella species (1 ml) alone or in combination with the diluted culture of E. coli (ATCC 202226) (1 ml) were seeded in ventilation-cup test tubes containing Nutrient Broth (5 ml). The tubes were incubated for 24 hours INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 APR 2007 REP.FIVFD at 37 °C. The number of colony-forming units (CFU) of the pathogens and of E. coli (ATCC 202226) was determined by plating the cultures on Nutrient agar. The CFU numbers of two Shigella species in the pure culture and in mixed cultures with both probiotic E. coli species are shown in Table 1 below.

Table 1 Probiotic Organism Growth of Shigella CFU/ml S. flexneri S. sonnel E. coli ATCC 202226 4 <5x10 <5x104 E. coli M-17 lxlO6 2xl06 Pure Shigella culture 6xl06 2x106 Example 8 Application of the food supplement (Example 4) and of Colibacterin (dry formulation of E. coli M-l 7) in hospitalized gastroenteritis patients A group of 60 patients that developed gastroenteritis following hospitalization was randomly divided into 3 sub-groups with a similar distribution of sex, age and the severeness of gasroenteritis symptoms. All patients received 15 normal supportive treatment, including rehydration, vitamins, etc. The severity of condition did not require treatment with antibiotics. The first group of 20 patients received 10 ml of the food supplement (Example 4) 3 times daily 30 minutes before the meals for 7 days. The second group of the same number received Colibacterin (Colibacterinum siccum) as recommended by the producer (one dose twice a day 20 30 minutes before the meal) and the third group received no supplement at all.

The onset of normalization (in days) of the symptoms of gastroenteritis in all groups were recorded and are shown in Table 2 below.

Table 2 Symptoms Onset of the normalization of the symptom, days Food supplement Colibacterin Control Body temperature 2.8±0.2 2.7±0.2 3.7±0.2 Intoxication 2.5±0.1 3.6±0.1 4.6±0.1 Abdominal pain 3.3±0.2 .1 ±0.2 6.1±0.2 Diarrhea 1.8±0.2 3.4±0.2 4.4±0.2 Table 2 (Continued) Intellectual property office of n.z. 17 APR 2DW RECEIVED- 21 Colibacterin (Colibacterinum siccum) is the freeze-dried preparation of live E. coli M-17 produced by BIOMED Ltd., Moscow, Russia, and recommended for use against diarrhea [Vidal Handbook: Pharmaceutical preparations in Russia (N.B. Nikolaeva, B.P. Alperovich and V.N. Sovinov, Eds.) AstraPharmService, 1997, 5 Moscow, p. 275].

Example 9 Application of the food supplement (Example 4) in patients with acute GI infections 10 Patients with severe GI infections of various etiologies: salmonellosis, escherichiosis, shigellosis, staphylococcal infections and food intoxications of unknown etiology were included in the study. In all patients the hospitalization was indicated by an acute onset of the disease and appearance of acute gastroenteritis. The total of 186 patients were treated with the food supplement and a similar group 15 of 102 patients received standard treatment.

The onset of normalization (in days) of the symptoms of gastroenteritis in all groups were recorded and are shown in Table 3 below.

Table 3 Symptoms Onset of normalization, days Study group Control Fever 1.2±0.3 2.7±0.3 Weakness 1.6±0.2 2.9±0.3 Abdominal pain 1.5±0.2 2.4±0.3 Diarrhea 2.3±0.2 4.7±0.4 Days in bed 4.8±0.3 6.7±0.4 In a separate study a group of 30 patients with intestinal typhoid was treated with the food supplement. In 80 % of patients the symptoms of disease disappeared within 3 days. Only in 3 cases the supplement treatment was stopped due to the development of more severe chronic colitis. 22 Example 10 Applications of the food supplement (Example 4) in patients with GI disorders caused by antibiotics Patients with severe GI disorders were divided in 2 groups. Group I 5 contained 48 patients with peptic ulcer disease who developed GI disorders after antibiotic treatment against H. pylori. Group II contained 22 patients that developed GI disorders following antibiotic treatment of pneumonia.

The food supplement (5 ml) was given 3 times a day before meals for 7 days. In both groups symptoms of diarrhea disappeared in 2-3 days in all the patients. 10 After the administration of the supplement, complete normalization of intestinal microflora was observed in 84.5 % of the patients. It was demonstrated by a dramatic increase in lactobacilli and Bifidobacteria, reduction on the general count of E. coli, complete disappearance of the hemolytic E. coli and other pathogens such as Staphylococci, Proteus vulgaris and even Candida sps. In the remaining 15 15.5 % of the patients, a significant improvement was observed.

Example 11 Application of the food supplement (Example 4) in patients with late radiation enterocolitis 20 The food supplement (10 ml, 3 times a day for 14 days, 30 minutes before the meals) was given to 24 patients with womb, colon and gastric cancer that developed enterocolitis following radiation therapy or a combination of radiation and chemotherapy.

Prior to the use of the food supplement, all patients complained about 25 frequent and painful desire to defecate, liquid stool (4-12 times a day) appearance of mucous (9 cases) and blood (3 cases) in the stool.

Two or three days after ingesting the supplement, all the patients noted the lessening of pain and reduction in the number of defecations; the stool had a normal appearance. Four to five days later the diarrheal syndrome was gone, the 30 appearance of blood and mucus ceased. The blood analysis showed a strong improvement in blood indicators.

In the control group of a similar size the symptoms persisted.

Example 12 Application of the food supplement (Example 4) in AIDS patients Patients suffering from AIDS frequently develop chronic diarrhea. A group of such patients was given 10 ml of the food MllecWATprc of n.z. 17 APR 2007 day RECEIVED 23 minutes before the meals for 20 days. The control group received no supplement. The results of the treatment are shown in Table 4 below.

Table 4 Parameter Food supplement Control No. of patients Average age, years 38±1 36±2 Daily defecation frequency: at the onset of the Exp. 3.4±0.3 3.6±0.3 at the end of the Exp. 1.1±0.1 3.2±0.3 one month after the Exp. 1.5±0.2 3.7±0.3 Av. onset of normalization 6.0±0.7 remained abnormal Patients receiving the food supplement showed normalization of intestinal microflora: reduction in general number of coliforms, disappearance of the hemolytic E. coli, increase in the numbers of Lactobacilli and Bifidobacteria, reduction in Candida sps.

Example 13 Application of the food/feed additive (Example 3) for accelerated weight increase in healthy piglets Healthy piglets were administrated 3 ml per os of the food/feed additive per piglet per day until weaning. The group receiving the feed additive gained weight at weaning on the average 1.0 kg per piglet more than the control group.

Example 14 Application of the feed additive (Example 5) in healthy piglets 20 Several hundreds healthy piglets were administrated 3 ml per os of the feed additive per piglet on the first and third day after delivery and at weaning. Mortality was cut down by 50 % compared with the control group receiving standard prophylactic treatment with antibiotics. The group receiving the feed additive gained weight at weaning on the average 0.39 kg per piglet more than the 25 control group. When the feed additive and antibiotic treatment were compared in the same litter, the weight gain in the piglets obtaining the feed additive was found higher by 2.4 kg than in the control. 24 Example 15 Application of the feed additive (Example 5) in piglets showing diarrhea Several hundred piglets showing diarrhea were given daily 5 ml per os of the feed additive per piglet. The control group of the same size was treated with 5 antibiotics: advocin, gentiamycin, amoxicillin. The symptoms of diarrhea in the group receiving the feed additive disappeared within 1-2 days. No mortality was observed, and piglets developed normally. Antibiotics stopped diarrhea in the great majority of piglets but the piglets remained stunted in their development.

About 70 retarded piglets, that received antibiotic treatment against diarrhea 10 for a week, and generally considered lost, were given the feed additive for three days. All but two survived.

Example 16 Application of the feed additive (Example 5) in healthy calves 15 Day-old healthy calves were administrated 5 ml of the feed additive a day in milk during 7 days. More than 95 % of the calves did not developed diarrhea until they were 14 days old, when a few cases positive for Rotavirus were diagnosed. Normally, the incidence of diarrhea in this farm is 20-30 %.

Example 17 Application of the feed additive (Example 5) in calves showing diarrhea Calves developing diarrhea were given daily 10 ml of the feed additive per animal with milk during 3-5 days. The symptoms of diarrhea disappeared within 1-2 days in 90-95 % of calves. In the remaining 5-10 % the diarrhea was caused by 25 virus. These calves were treated with antibiotics with poor results.

Example 18 Application of the feed additive (Example 5) in healthy lambs and goat kids Day-old healthy lambs and goat kids were administrated 3 ml of the feed 30 additive a day in milk during 7 days. In some cases slight to moderate signs of diarrhea were observed. These signs usually disappeared spontaneously or were successfully treated with an increased dose of the feed additive (5 ml). Normally the insidence of diarrhea in these farms is 50 % for baby lambs and for goat kids. intellectual property office OF N.Z. 17 APR 2007 i received.! Example 19 Application of the feed additive (Example 5) in lambs and goat kids showing diarrhea The feed additive was tested in a herd suffering from pathogenic E. coli 5 infections. In the year preceding the experiment, about 90 from 120 lambs and goat kids died from diarrhea. Treatment with antibiotics was ineffective, since the disease developed suddenly and with fast mortality. Lambs and goat kids developing diarrhea were given daily 5 ml of the feed additive per animal with milk during 3-5 days. A matching group of animals was treated, as normally 10 recommended, with antibiotics. The symptoms of diarrhea disappeared within 1-2 days in about 90 % of the lambs and goat kids receiving the feed additive. Their further development appeared normal. The control group receiving antibiotics (gentamycin) showed much poorer results. Diarrhea in this group persisted, calling for repeated treatment with antibiotics. The development of the control group was 15 severely retarded.

Example 20 Application of the feed additive (Example 5) in poultry The feed additive was added to the drinking water with the average uptake 20 of 0.01 ml of the additive per day per chick during the breeding period (42-49 days). An increase of 3.2 % in weight gain, accompanied by 4 % improvement in food conversion was noted in controlled trials in broilers.

Excellent results were also obtained with turkeys of age 1 day to 6 weeks. Each bird received 0.01 ml of the additive per day. A weight gain of over 10 % 25 was observed, along with reduced mortality rate. Birds which still exhibited diarrhea, were treated with 0.1 ml per day of the food additive, without any treatment by antibiotics, and showed better recovery from the control birds which were treated with only antibiotics.

Example 21 Application of the feed additive (Example 5) in dogs and cats Application of the feed additives in puppies resulted in cessation of the symptoms of diarrhea within 24-48 hours.

INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 APR 2007 RECEIVED 26 Example 22 Application of the feed additive (Example 5) in mice; Evidence for immunostimulation Two-weeks-old C571B1 mice were divided into 4 groups of 10 animals in each. Mice were deprived water from 4:00 pm to 8:00 am and then given 15 ml of either water (groups 1 & 3) or the feed additive (groups 2 & 4). This treatment was repeated for 2 weeks. At the end of this treatment mice from groups 3 and 4 were subjected to laparotomy under ether narcosis. A cut of 1.5 cm was made in the abdominal wall. Irritation of intestine was carried out by a dry gause. All the mice were sacrificed 24 hours after laparatomy; spleens were excised. Splenocytes were isolated and cultured on RPMI medium for 48-72 hours in the presence of phytohemagglutinin (PHA). In the group treated with the feed additive the proliferative activity of splenocytes reduced by laparotomy was recovered.

Splenocytes proliferation induced by PHA: 1. Control group/water 3060±290 2. Control group/water/laparotomy 2120±300 3. Test group/BioCocktail 2740±370 4. Test group/BioCocktail/laparotomy 3040±520 Example 23 Application of the food additive (Example 4) in human; Evidence for immunostimulation A number of patients were operated for malignant tumors of womb, cervix, stomach, and intestine. The gynecological patients (20 patients) received follow-up radiotherapy; the abdominal patients (30 patients) received chemotherapy. About half of the patients received the food additive twice daily for the duration of therapy. In all patients receiving the food additive there was observed a significant immunostimulation, as expressed in: T-lymphocytes (CD3+, HLA-, DR+) Lymphocyte blast transformation Gynecological tumors Abdominal tumors Control Food Additive Control Food Additive 0.03±0.01 0.10±0.03 1.0*0.1 1.6±0.2 INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 APR 2007 received 27 a. spontaneous 530±110 b. PHA-induced 1600±360 Phagocytes No. (%) 60.7±2.9 880±80 700±120 5720±2100 4400±1340 76.5±2.2 57.7±1.5 1070±100 13370±3720 74.2±l .9 Example 24 Bacteria preservation effect of the formulation E. coli cells were dispersed in a formulation prepared as described under 5 Example 1 above and was maintained for 6 months at room temperature. Both at the beginning and at the end of the 6 months period, samples derived from the dispersion were seeded at appropriate consecutive dilutions on nutrient broth agar plates. The number of colony forming units was determined for both samples. The concentration determined was 1x10 in both cases, showing the effect of the 10 formulation in preserving microorganisms. The above experiment was performed in parallel with Pseudomonas putida, yielding the same results.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and 15 variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual 20 publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 APR 2007 received

Claims (17)

28 WHAT IS CLAIMED IS:

1. The use of a formulation comprising at least one volatile fraction (VF) of a plant extract, said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C; to sustain viability of as much as 107 cells/ml of at least one microorganism for at least two months at room temperature, with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).

2. The use of claim 1, wherein said at least one microorganism is selected from the group consisting of Escherichia spp., Alcaligenes spp., Arthrobacter spp., Bifidobacterium spp., Lactobacillus spp., Lactococcus spp., Nitrosomonas spp. and Pseudomonas spp.

3. A dispenser of microorganisms comprising a reservoir and a dispensing mechanism being connected thereto, said reservoir housing at least one microorganism, in a formulation containing at least one volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction is in an effective amount to sustain viability of said at least one microorganism for at least two months at room temperature, with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).

4. The dispenser of claim 3, wherein said dispenser is airborne.

5. The dispenser of claim 3, wherein said dispenser is hand held. fPONZ 14 JAh 2a)8 29

6. The dispenser of claim 3, wherein said dispenser is designed to automatically dispense a predefined volume.

7. The dispenser of claim 6, wherein said dispenser is designed to automatically dispense said predefined volume periodically.

8. A method of biocontrol of an organism, the method comprising the step of spreading in an endemic area at least one viable microorganism capable of biocontrolling the organism, said at least one viable microorganism being in a formulation containing at least one volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction is selected so as to sustain viability of as much as 107 cells/ml of said at least one microorganism for at least two months at room temperature, with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).

9. A method of eliminating an oil pollution, the method comprising the step of spreading in a polluted area at least one microorganism capable of degrading oil, said at least one viable microorganism being in a formulation containing at least one volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction is selected so as to sustain viability of as much as 10 cells/ml of said at least one microorganism for at least two months at room temperature, 1* J AH 2Q08;I;30;with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).;

10. A method of restoring organic fumes degrading microorganism population in a biofilter, the method comprising dispensing onto the filter at least one microorganism capable of degrading organic fumes into environmentally harmless compounds, said at least one microorganism being in a formulation containing at least one volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction is selected so as to sustain viability of as much as 107 cells/ml of said at least one microorganism for at least two months at room temperature,;with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).;

11. A method of preparing a starter for a fermentation process, the method comprising growing an amount of a starter microorganism sufficient to induce a fermentation process, and suspending said starter microorganism in a formulation containing at least one volatile fraction (VF) of a plant extract, wherein said plant extract is obtained by water extraction and said volatile fraction being prepared by steam distillation of said plant extract under a pressure lower than atmospheric pressure and at a bath temperature not exceeding 38 °C, said at least one volatile fraction is selected so as to sustain viability of as much as 107 cells/ml of said at least one microorganism for at least two months at room temperature,;with the proviso that said microorganism is not Escherichia coli strain BU-230-98 ATCC Deposit No. 202226 (DSM 12799).;IPONZ;i 1 * JAN 2008 31

12. A use according to claim 1, substantially as herein described or exemplified.

13. A dispenser according to claim 3, substantially as herein described or exemplified.

14. A method according to claim 8, substantially as herein described or exemplified.

15. A method according to claim 9, substantially as herein described or exemplified.

16. A method according to claim 10, substantially as herein described or exemplified.

17. A method according to claim 11, substantially as herein described or exemplified. THE BIO BALANCE CORP. By Their Attorneys HENRY HUGHES per- IPONZ 1A JAt-. 2008