RU2802308C2 - Compositions of volatile organic substances with antimicrobial activity - Google Patents

Abstract

FIELD: medicine; chemistry.

SUBSTANCE: group of inventions relates to new chemical compositions with antimicrobial activity, and methods for their use. An antimicrobial composition suitable for use directly or as a component in other compositions, having antimicrobial activity and containing a combination of isoamylhexanoate and at least one acid selected from propanoic acid and isobutyric acid, and an acceptable carrier are proposed. The antimicrobial composition can be used in cleaning and pharmaceutical compositions, as well as in a method of treating a human or animal having a microbial infection.

EFFECT: inventions provide compositions with improved antimicrobial activity.

24 cl, 28 dwg, 12 tbl, 19 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority over U.S. Provisional Application No. 61/842,362, filed July 2, 2013, and U.S. Provisional Application No. 61/948,902, filed March 6, 2014, each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The importance of safely disposing of billions of pounds of human and animal excreta every day in a way that avoids the many health problems associated with such waste cannot be overemphasized. In reality, only a fraction of the huge amount of excreta is safely disposed of, while the rest is untreated waste and poses a threat to human and animal health. For example, it is well known that a combination of bacterial and other agents that cause gastrointestinal disease is the leading cause of death in the world. In addition, it is well known that these types of diseases primarily affect newborns and children, as well as livestock. It is estimated that over the next ten years at least twenty million people will die as a result of pollution and poor sanitation.

One reason for this is that some 2.4 billion people live in areas that lack proper sanitation. Nearly 4,000 children die every day from diseases such as diarrhea. In addition, people who suffer from waterborne diseases occupy about half of the world's hospital beds. In several Asian countries, diarrhea-related diseases kill twice as many people as AIDS. Essentially, non-sanitary conditions related to the inability in homes, communities and, in some cases, entire countries, to adequately handle and dispose of human and animal excreta that stimulate the growth and development of pathogens.

Undoubtedly, the undesirable effects of microorganisms in industrial applications are numerous. For example, safer and more effective products are needed to clean germ-contaminated surfaces in healthcare facilities. Safer and more effective means are needed to treat crops against unwanted microbial growth. Further, a means is urgently needed to reduce the undesirable odors produced by the breakdown of excrement in industrial farming.

There is an urgent need to replace antibiotics with other types of compounds that also have antimicrobial activity. The continued use of the most commonly used antibiotics in livestock and agriculture has led to acquired resistance in microbial populations, especially microbes that can be pathogenic. Every year, at least 23,000 people in the US die due to infections caused by drug-resistant bacteria, and the number is growing.

Thus, there is a need in the art for antimicrobial compositions suitable for reducing the number of microorganisms and reducing the impact of microbial products in a wide range of industrial applications, as well as for preparations and methods for treating human and animal excrement. The present invention satisfies this need.

BRIEF DESCRIPTION OF THE INVENTION

In one embodiment, the present invention relates to a chemical composition having antimicrobial activity containing propanoic acid, isobutyric acid and at least one ester. In another embodiment of the invention, at least one ester is isoamyl hexanoate. In another embodiment of the invention, the composition further comprises at least one carrier, which is selected from the group consisting of bentonite, zeolite and perlite. In another embodiment, the ratio of propanoic acid: isobutyric acid: isoamyl hexanoate is about 3.5:3.5:2 v/v/v. In another embodiment of the invention, the ratio of propanoic acid, isobutyric acid and isoamyl hexanoate is about 7 parts of two acids and 2 parts of isoamyl butyrate. In another embodiment of the invention, the composition further comprises at least one endophyte. In another embodiment, the endophyte belongs to the genus Fusarium .

In another embodiment, the present invention relates to a chemical composition comprising predominantly propanoic acid, isobutyric acid, isoamyl hexonoate and a carrier selected from the group consisting of bentonite, zeolite and perlite.

In another embodiment, the present invention relates to a chemical composition containing propanoic acid and at least an ester containing an acidic residue with 6-12 carbon atoms, and the chemical composition has a propanoic acid:ester ratio of approximately 7:2 v/v. In another embodiment of the invention, at least one ester is isoamyl butyrate. In another embodiment of the invention, the composition further comprises at least one nutritional supplement and at least one salt. In another embodiment of the invention, the composition contains glucose, whey protein, potassium chloride, magnesium sulfate and sodium chloride. In another embodiment of the invention, the composition contains glucose, glycine, potassium chloride, sodium chloride and magnesium acetate. In another embodiment, the composition contains glucose, glycine, potassium chloride, sodium chloride, magnesium acetate, and potassium dihydrogen phosphate. In another embodiment of the invention, the composition further comprises at least one carrier. In another embodiment, the composition contains predominantly propanoic acid and isoamyl hexonoate in a propanoic acid:isoamyl hexonoate ratio of about 7:2 v/v. In another embodiment of the invention, the composition contains at least one endophyte. In another embodiment, the endophyte belongs to the genus Fusarium .

In another embodiment, the invention relates to a method of treating an animal having a disease or disorder associated with a microbial infection, which comprises administering to the animal an effective amount of a composition comprising at least one organic acid and at least one ester. In another embodiment of the invention, the present invention relates to a composition that contains propanoic acid and at least one ester containing an acidic residue with 6-12 carbon atoms, and the chemical composition has a propanoic acid: ester ratio of about 7:2 v/v .

BRIEF DESCRIPTION OF GRAPHICS

The following detailed description of embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, which is illustrated in the drawings of the presently preferred embodiments of the invention. It should be understood, however, that the present invention is not limited to the precise arrangements and means in the embodiments of the invention shown in the drawings.

Fig. 1 illustrates a petri dish for biotesting to determine the bioactivity of various esters in a 1:1 mixture of two organic acids according to Table 2. Test organisms were as follows - Cercospora (dark bottom left bottom) then clockwise - Phytophthora , Verticillium , Sclerotinia , Pythium , Fusarium , Trichoderma , Rhizoctonia , and Aspergillus . Strokes - Saccharomyces (bottom far right) then Candida , E coli and Bacillus (bottom left). A=control petri dish and B=petri dish with System 1 after incubation for 30 hours.

Fig. 2 illustrates the effect of System 1 on bacterial growth in human feces. 5 mg of fresh human feces was spread over the surface of a Petri dish with potato dextrose agar. The plugs were then removed from the center and the bentonite was placed in about 0.5 g wells. The bentonite in the center well contained no System 1 ingredients, but the well to the right of the center had System 1 in a volume of 1 ml of System 1 per 10 g of bentonite. Petri dishes were incubated for 48 hours at 22°C and then photographed. Bacterial growth was not detected in the Petri dish treated with System 1, but there were sufficient bacterial colonies in the control Petri dish.

Fig. 3 illustrates the effect of System 2 on bacterial growth in human feces. 5 mg of fresh human feces was spread over the surface of a potato dextrose agar petri dish. The plugs were then removed from the center and the bentonite was placed in about 0.5 g wells. The bentonite in the center well contained no System 1 ingredients, but the well to the right of the center had System 1 in a volume of 1 ml of System 1 per 10 g of bentonite. Petri dishes were incubated for 48 hours at 22°C and then photographed. Bacterial growth in Petri dishes treated with System 2 was not detected.

Fig. 4 illustrates two cat litter boxes with cat feces each from 5 different cats about 140 gr. The container on the right was treated with System 1 on bentonite (0.5 ml/100 g bentonite). After 5 days ammonia measurements showed 14 ppm in the control container (left) and 0 ppm in the treated container. Overall, the odor was significantly reduced in the treated container.

Fig. 5 illustrates the treatment of about 140 g of human feces with urine with Fusarium subglutinans 06-1 in the presence of System 2 (1 ml per 10 g of zeolite). After 3 weeks there was a significant growth of Fusarium subglutinans (white mycelium in the right container). The ammonia level was 71.4 in the control container on the left and 12.1 in the treated container on the right. No fungal growth and no degradation of excrement occurred in the control variant of the experiment (left).

Fig. 6 illustrates the progressive growth of Fusarium spp on small pieces of human feces weighing about 100 mg (wet weight) over many days. Newly growing Fusarium spp. were isolated and characterized. other than P2-24 ( Fusarium culmorum ). New Fusarium spp. especially E06-1 and E06-5 grow faster on human feces. Growth was measured by the radius of mycelium removal from an agar block placed on a piece of human excrement.

Fig. 7 illustrates a six-day culture of the fungus Fusarium subglutinans E06-1 (top), which is preferably used to treat human and animal faeces in combination with System 2. Light microscopy of spores and hyphae F. subglutinans (bottom). The spores are slightly curved and have 9.8 -12×2.5 µm.

Fig. 8 illustrates the vigorous growth of Fusarium subglutinans (E06-8) on human feces (centre) in the presence of System 2 with bentonite as a carrier. Note the inhibition of bacterial growth on the left and center of the culture dish, which is influenced by System 2 fumes from the bentonite particles on the left side of the dish, allowing the fungus to grow. There was added 0.5 g of treated bentonite, about 100 mg of human excrement, Petri dishes were incubated for 12 days. See fig. 6 with comparative height measurements.

Fig. 9 illustrates the biological activity of various test mixtures against the background of test microorganisms. A small fragment of each organism was placed in the periphery of the CDA in a Petri dish. The test solution was placed in the central well in a plastic cup holder. The control dish (A) was also arranged. After 30 hours of growth of test organisms compared to control, % inhibition was calculated. The Petri dish (B) contained the test mixture. 30 hours after the Petri dish was placed, measurements were taken.

Fig. 10 illustrates the reduction in microbial contamination of crushed grain 1 hour after treatment at various concentrations of C-3 solution. Concentrations above 0.5% completely eliminated bacterial contamination as seen from the absence of bacterial colonies at 0.5% and 1.0% treatments (see above). Some minimal fungal contamination was observed in the last variant - note the two fungal colonies on each of the cups on the right. Incubated for two days at room temperature and then photographed.

Fig. 11 illustrates the use of bentonite with various pretreatment compositions (C) for 3 days to kill E. coli in human feces (stripe in the middle of the dish) and at the same time ensure the growth of fusarium (top of the dish), which otherwise would not allow the consumption of solids in human feces.

Fig. 12 illustrates the effectiveness of formulation C-3 in treating chicken feces, first by applying the suspension to PDA plates and then by adding 0.5 g of zeolite treated with 3 ml of C-3 per pound (453 g) of vehicle. The photo was taken after 3 days of incubation at room temperature. It can be seen that the Petri dish containing C-3 zeolite was practically free from bacterial contamination.

Fig. 13 illustrates 1 ft ^-2 plastic containers tightly closed at the top, filled with treated manure with the addition of untreated bentonite in the proportion indicated in the instructions on the package, to compare the effectiveness of the composition.

Fig. 14 illustrates the average ammonia level obtained in a 5 minute interval every 24 hours (A). Peak ammonia levels follow a similar trend with C-1 treated litter, showing a significant reduction in ammonia levels (B). Fig. 14B illustrates the peak level of ammonia taken from the study 5-minute interval every 24 hours.

Fig. 15 illustrates the microbiological activity of C-1 compared to a control. Figures 15A and 15B show that the control bentonite (15A) had a huge amount of bacterial colonies growing in the cup, including those areas close to the well containing the manure. In contrast, C-1 treatment (4 ml C-3 per pound (453 g) vehicle, 15B) was substantially free of bacterial colonies around the well in the Petri dish.

Fig. 16 illustrates 1 ft ^-2 plastic containers tightly closed at the top, filled with pine shavings and bedding recommended to be processed in the proportion indicated on the instructions on the package. For these tests, variants were tested using C-1 at 15 ml per pound (453 g) of zeolite and a control with untreated zeolite.

Fig. 17A illustrates the average ammonia level obtained in a 5 minute interval every 24 hours. The ammonia peak shows a similar trend with Barnyard Bedding showing the lowest ammonia production levels (FIG. 17B). Fig. 17B illustrates the peak level of ammonia taken from the investigated 5-minute interval every 24 hours.

Fig. 18 illustrates 1 ft ^-2 plastic containers tightly closed at the top, filled with pine shavings (a common bedding material for large animals) and bedding treated in the recommended proportion indicated on the packaging instructions. For these tests, a control variant with untreated zeolite and a variant with treated C-1 were tested.

Fig. 19 illustrates the average ammonia level obtained in a 5 minute interval every 24 hours (19A). Peak ammonia levels follow a similar trend with C-1 treated bedding, showing the lowest levels of ammonia production (19B). Fig. 19B illustrates the peak level of ammonia taken from the investigated 5-minute interval every 24 hours.

Fig. 20 illustrates diarrhea in a calf before treatment with C-X solution (FIG. 20A), and after two courses of treatment with C-X solution (FIG. 20B).

Fig. 21 illustrates diarrhea in a calf before treatment with C-X solution (FIG. 21A), and 24 hours after treatment with C-X solution (FIG. 21B).

Fig. 22 illustrates the condition of dairy cows in barn 1.

Fig. 23 illustrates the typical creamy yellow diarrhea seen in calf 919 (FIG. 23A) and calf 919 fully recovered after treatment with C-X solution (FIG. 23B).

Fig. 24 illustrates a calf 166 at ranch 9 suffering from diarrhea during the winter of 2014 (FIG. 24A) and after one day of treatment with C-X solution (FIG. 24B), where the animal recovered and the yellow diarrhea subsided.

Fig. 25 illustrates sheep with mastitis (FIG. 25A) and administration of the C-3 composition to the animal using a syringe (FIG. 25B).

Fig. 26 illustrates raspberries treated with bentonite (control) in the center well (FIG. 26A) and those treated with C-3 1:10 (FIG. 26B) and stored for 1 week at room temperature. Berries treated with C-3 remained edible and did not rot.

Fig. 27, includes figures 27A-27B, illustrates soil treated with P. ultimatum or C-3. Fig. 27A is a photograph of P. ultimatum treated soil with red beet seeds. Germination of only one or two seeds was observed. Fig. 27B is a photograph of soil treated with C-3 on bentonite in the presence of P. ultimatum and red beet seeds. Germination of many seeds was observed.

Fig. 28, includes Figures 28A-28D, illustrates images of an aqueous solution of agar-agar in Petri dishes for test C-3 with red beet seeds. Figure 28A is a depiction of a Petri dish with medium and red beet seeds, bentonite, C-3 (1 part to 10 g bentonite), and P. ultimum . The influence of C-3 on the growth of P. ultimum cells was established. Fig. 28B shows a Petri dish with medium and red beet and P. ultimum seeds. Fig. 28B is an image of a petri dish with medium and red beet seeds. Fig. 28 D is an image of a medium petri dish, demonstrating that C-3 was not harmful to red beet seeds.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant to a clear understanding of the present invention, eliminating, for purposes of clarity, many other elements found in typical antimicrobial formulations. Those skilled in the art may recognize that other elements and/or steps are desirable and/or necessary to carry out the present invention. However, since such elements and steps are well known in the art, and because they do not contribute to a better understanding of the present invention, such elements and steps are not discussed in this document. The present description herein is directed to all such variations and modifications, as elements and methods, known to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they are commonly understood by any person skilled in the art to which this invention belongs. While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described.

As used in this document, each of the following terms has the meaning associated with it in this section.

The articles ʺAʺ and ʺanʺ are used herein to refer to one or more than one (i.e., at least one) grammatical entity of an entry. For example, ʺelementʺ means one element or more than one element.

“About”, used herein when referring to measurable quantities such as quantity, time period, and the like, is intended to cover variations of ±20%, ±10%, ±5%, ±1%, and ±0.1% of the set value. , since such variations are suitable.

"C-1" as used herein refers to any and all formulations of System 1.

"C-2" as used herein refers to any and all compositions of System 2.

"C-3" as used herein refers to any and all compositions of System 3.

"C-4" as used herein refers to any and all compositions of System 4.

"C-5" as used herein refers to any and all compositions of System 5.

"C-X" as used herein refers to any and all compositions of System X, which may contain one or more of Systems 1-5.

Used in this document, the term "CLOE" means a composition containing C-1 or C-5.

Used in this document, the term "Barnyard Bedding" means a composition containing C-1 or C-5.

As used herein, the term "pharmaceutical composition" refers to a mixture of at least one composition in accordance with the present invention with other chemical components such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickeners and/or excipients. The pharmaceutical composition facilitates the introduction of the composition into the body. Several routes of administration of the composition exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

As used herein, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not interfere with the biological activity or properties of the composition and is relatively non-toxic, i. from the components of the composition in which it is contained.

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or carrier, such as a liquid or solid excipient, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickener, solvent, or encapsulating agent, appropriately involved in the transfer or transport of the composition. within the scope of the present invention or to the patient so that he can perform his functions, Typically, such constructs are produced or transported from one organ or body part to another organ or body part. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition, including the usefulness of the composition in the present invention, and harmlessness to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffer substances such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, a "pharmaceutically acceptable carrier" contains any antibacterial and antifungal agents, absorption delaying agents, and the like that are compatible with the activity of the composition used in this invention and physiologically acceptable to the patient. The compositions may contain additional active compositions. A "pharmaceutically acceptable carrier" may further comprise a pharmaceutically acceptable salt according to the present composition useful in the present invention. Other additional ingredients that may be contained in a pharmaceutical composition used in the practice of this invention are known in the art, and are described, for example, in "Remington Pharmaceutical Sciences" (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA ), which is incorporated herein by reference.

In this specification, various aspects of the present invention may be represented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a permanent limitation on the scope of the present invention.

Accordingly, the description of the range should be considered, namely, to disclose all possible subranges, as well as individual numerical values within this range. For example, in a range description, 1 to 6 should be considered to have the described subranges, namely, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, 6 in any integer or fractional step in between. This applies regardless of the latitude of the range.

The present invention relates to the discovery of effective and useful chemical compositions, alone or in combination with certain endophytic fungi such as Fusarium spp, which have strong antimicrobial activity and can be used for various purposes such as: reducing microbial growth on surfaces of medical equipment or instruments, reducing microbial growth on agricultural plants, or decontaminating, degrading and deodorizing human and animal excrement. For example, the compositions of the present invention are suitable for waste treatment in any location, such as latrines, cat litter, pet stores, barns, chicken coops, pigsties, households, zoos, and a variety of other places.

In a preferred embodiment of the invention, an appropriate combination of harmless compositions containing ingredients that are on the list of substances recognized as completely harmless by the Food and Drug Administration (USA) and, accordingly, Fusarium spp endophytic fungi, such as F. subglutinans, are placed together in container, such as a biodegradable plastic bag. In addition, the container contains the required amount of a urine-absorbing polymer that is compatible with the endophyte F. subglutinans. This combination of active substances is a safe, fast and modern way to process substances found in human and animal excrement. Having these two ingredients in the bag effectively kills many of the harmful bacteria in human feces and at the same time begins the process of converting organic waste constituents into harmless soil additives.

The present invention can be applied in connection with national emergencies, military maneuvers, maritime activities, natural disasters, outdoor sports (camping, hiking, canoeing, hunting, cycling, etc.) and other activities in which human excrement must be properly and safely disposed of. This also applies to the development of safer products for all types of farm animals and pets.

As an example, it has recently been noted that the correct and safe disposal of human excreta is an important issue in the sound management of undeveloped areas of the globe. Aesthetics, as well as health issues, are the main issues facing the leaders of these territories. Accordingly, the present invention may be suitable for human and animal coverings, plant coverings, industrial surfaces, machine tools, and a variety of other uses.

In one embodiment, the compositions of System 1 (C-1), System 2 (C-2), System 3 (C-3), System 4 (C-4), and/or System 5 (C-5), and optionally the use of bentonite, zeolite or perlite as a carrier (depending on the application) are combined together in a container and the processes of bacteria killing and/or waste degradation begin immediately. In another embodiment, the present invention can be used in animal bedding and stall treatment, wherein the chemical mixture (with carrier) can be used directly in the animal's habitat, resulting in almost immediate killing of bacteria that cause harmful odors, such as ammonia. In another embodiment, the present invention may be applied to surfaces, such as crop covers, surfaces of medical equipment, medical or industrial tools, or the like, to eliminate or otherwise reduce the number of microorganisms on the treated surface.

Antibiotics are substances that kill or inhibit the growth of bacteria. A common misconception is that antibiotics are effective against other microorganisms, such as fungi and viruses, when in fact antifungal and antiviral compounds are needed for such purposes. Antibiotics work by interfering with key steps in the metabolism and reproduction of bacteria, and can be grouped into two main categories: bactericidal and bacteriostatic, depending on whether they kill bacteria or simply inhibit their growth, respectively. Antibiotics are generally safe for human use because they are either specific to certain types of bacteria or effective against bacteria at very low concentrations that are considered safe for humans. Other classes of chemicals, such as certain alcohols, acids, and peroxides, may have a wide range of inhibitory and/or damaging capabilities as they affect fundamental biochemistry common to many life forms. These types of compounds are classified as antiseptics, sterilizing, disinfecting and disinfecting agents, preservatives and depending on the specifics of their impact on the life of microorganisms, modes of effective use and toxicity to humans. Systems according to the present invention, such as C-1, are mixtures consisting predominantly of short chain organic acids and esters, in particular propanoic acid and isoamyl hexanoate. Neither of these molecules is classified as an antibiotic, but both have antimicrobial properties and can be either bactericidal or bacteriostatic depending on concentration and duration of use.

The systems according to the present invention do not work by the same mechanisms as antibiotics. While antibiotics target very specific steps, often by recognizing very specific structural moieties, the systems of the present invention kill bacteria and inhibit their growth by affecting fundamental biochemical properties necessary to sustain life. In addition, the components of the system in accordance with the present invention act synergistically so that the effect of the total mixture is greater than the sum of its parts. The mechanism for the synergistic effect observed with these systems is not understood, but other acid/ester mixtures show the same slightly increased combined effect.

One of the main components of the systems of the present invention is propanoic acid, a short chain organic acid that has established itself in use as a preservative in the food and agricultural industries. Most organisms, including humans and many bacterial species, have metabolic pathways that facilitate the use of propanoic acid as a nutrient, and indeed, one group of bacteria can produce these molecules. Thus, low concentrations of propanoic acid are, in fact, harmless to almost all organisms, but at high concentrations, it cannot be rapidly degraded and begins to accumulate inside the cell. With an increase in its concentration inside the cell, the acidity inside the cells also increases. When the acidity inside the cell is too high, the enzymes cannot function properly, DNA and other biological molecules are destroyed, and the cells die. Recent studies show that although the effect on intracellular acidity is the main antimicrobial mechanism of weak organic acids, it is by no means the only mechanism. When acids dissociate and release protons inside the cell, they become negatively charged. A high concentration of negatively charged molecules within a cell has many detrimental effects associated with osmotic pressure, nutrient storage, and metabolism.

At lower concentrations, acids can be inhibitory but not lethal. An increase in acidity occurs when an acid dissociates and releases a proton. When the acidity inside the cell rises significantly, the cell can transport protons outward in an attempt to maintain a normal pH level. Although effective, this strategy requires a large amount of energy and is non-lethal only at low acid concentrations. Because small organisms are more sensitive to smaller amounts of propanoic acid, concentrations that are harmless to humans can be lethal or inhibitory to bacteria. Propanic acid is not the only organic acid in C-1, but other similarly sized organic acids have antimicrobial activity, presumably occurring through essentially the same mechanisms.

The antimicrobial mechanism of esters remains largely unknown. Without wishing to be bound by any theory, one possible clue comes from the fact that for a given set of esters, those that are able to integrate more efficiently into the bacterial cell membrane tend to have an enhanced antimicrobial effect. The incorporation of any molecule into the cell membrane changes the chemical and physical properties of the membrane, resulting in changes in nutrient uptake, excretion, energy generation, and other vital cellular processes. While not wishing to be bound by any theory, this observation has led to the suggestion that the incorporation of certain esters into the cell membrane alters the chemical and physical properties in ways that adversely affect the organism. Cell membrane alteration is also the mechanism by which longer chain organic acids are thought to be affected.

Once antibiotics began to be used on a massive scale during the 20th century, the problem of antibiotic resistance became a serious clinical problem. In the 21st century, as the effects of antibiotic resistance became more visible and widespread, the concept entered the public consciousness and was finally recognized as a serious problem. In a bacterial population that is exposed to antibiotics, resistance either exists in a very small number of individuals or initially arises due to natural mutation and subsequently survives as antibiotic-resistant individuals have a survival advantage over non-resistant individuals. Antibiotic resistance spreads both vertically, from resistant cells to their offspring, and horizontally (direct transfer of resistance genes from resistant cells to non-resistant cells). Thus, resistance is rapidly spreading and antibiotic use is increasing, which is exerting selective pressure to increase the survival advantage of antibiotic resistance.

Bacteria can acquire resistance to a given antibiotic through four main mechanisms: they improve the enzymes that inactivate the antibiotic, change the structure of the target so that the antibiotic can no longer bind to the target, change the metabolic pathways to neutralize the inhibitory effect of the antibiotic, and develop an efflux pump that pumps out the antibiotic. outside the cell. Each mechanism has a genetic basis and can thus be transmitted from a cell that initially developed resistance to non-resistant cells. In some cases, bacterial cells can become resistant to various types of antibiotics. These are the so-called 'superbugs' that are emerging as the use of antibiotics increases in the agricultural, veterinary and medical industries, so multidrug-resistant bacterial strains will increase. In addition, combinations of small organic molecules such as acids and esters, which act in a synergistic manner, have been found to have virtually the same antimicrobial effect as antibiotics. Organic molecules that have these properties are referred to as "synergists".

The considered mechanisms of action of antibiotics, as well as resistance to them, can be summarized in one word - specificity. Antibiotics work by targeting specific structural features, enzymes, and macromolecules. In addition, antibiotic resistance occurs when bacteria develop an antibiotic-specific efflux pump or alter specific structural features, enzymes, or macromolecules. Where the antibiotics are specific, the components of the systems of the present invention are general. Organic acids and esters do not have specific targets; instead, they exert their antimicrobial effects by altering the biochemical environment of the bacterial cell. They are effective against a much wider range of organisms and they interfere with many cellular processes.

Organic acids are widely distributed in nature. Any bacterial cell is invariably exposed to organic acids at some point in its life, and as a result, many bacterial species have innate genetic mechanisms that, by induction, help them cope with the stresses caused by natural organic acid exposure. Perhaps the most studied is Salmonella's acid tolerance. Essentially, when a salmonella cell exposed to a high, but sub-lethal, concentration of acid, it induces the expression of a number of genes such that the next time it is exposed to an acidic environment, its chances of survival are much higher. This has been demonstrated experimentally: by acid culture of previously acid-treated and non-acid-treated Salmonella cells. In almost every case, previously treated cells showed a much higher resistance to acid.E. coli acid tolerance has also been extensively studied and it is likely that this mechanism is present in many other bacterial species. In the case of pathogenic microbial species like E. coli and Salmonella, there is great concern that the induction of acid resistance by exposure to sub-lethal concentrations of organic acids in food preservatives may increase bacterial virulence because bacteria have a better chance of surviving acidic gastric fluid during digestion. .

However, there is an important difference between antibiotic resistance and organic acid resistance. Many antibiotic resistance genes are located on mobile genetic elements known as plasmids, which are easily transferred between bacterial cells and bacterial species. Thus, the acquisition of resistance to numerous antibiotics is a relatively simple process for any bacterial cell. The mechanism of acid resistance, such as acid tolerance, on the other hand, is encoded on chromosomal DNA. This genetic information can only be passed on to the cell's progeny, and thus the unexpected appearance of "superbugs" that occurs with multidrug-resistant bacterial strains is not observed here. Moreover, without wishing to be bound by any theory, it is possible that the synergistic effect observed when using an organic acid in combination with organic esters may negate some of the mechanisms of acid resistance.

In one embodiment of the invention, in order to identify a microorganism that can grow on human or animal feces and as a result destroy them, it is necessary to develop a new antibacterial mixture to kill the microorganisms contained in the waste, which normally break down urea to ammonia and uric acid. Ammonia is lethal to most fungi, which otherwise degrade the solid constituents of the waste. The basis of this discovery is the known fact that propanoic acid has antibacterial properties, but only to the level of inhibition, and the same is true for isobutyric acid. Thus, these two compounds were the starting ingredients for new and effective antibacterial mixtures. An additional ingredient was required in the mixture, which has a lethal effect on microorganisms. Then, in a completely unexpected way, it became known that the addition of certain esters to these small organic acids, gave them a significant improvement in antimicrobial activity.

Microorganisms living in the world's rainforests, in order to survive, must constantly develop biochemical mechanisms to cope with potential competitors. In this regard, they have developed the ability to produce antimicrobial molecules and compounds that inhibit and destroy other microorganisms. As new antibiotics are in demand by mankind, researchers will visit the rainforests in search of new microorganisms and substances that they produce to suppress and destroy other microbial competitors. Several rainforest microorganisms provided important chemical clues that allowed the selection of compounds for systems 1-4.

Compositions

In part, the present invention includes a chemical composition containing at least one organic acid, such as propanoic acid, isobutyric acid or butyric acid. In one embodiment, the chemical composition has antibacterial activity when applied to human or animal feces. In some embodiments of the invention, the organic acids used may contain from 2 to 5 carbon atoms, and the content of each acid may vary from 0% to 80% of the bioactive mixture. In a preferred embodiment of the invention, the organic acid is propanoic acid. In another embodiment of the invention, the present invention includes a chemical composition consisting predominantly of organic acids, such as propanoic acid, isobutyric acid or butyric acid. In one embodiment of the invention, the chemical composition contains predominantly propanoic acid. In some embodiments of the invention, the chemical composition contains two organic acids. In one embodiment, the two organic acids are propanoic and isobutyric acids. In one embodiment of the invention, the chemical composition contains a combination of two organic acids and at least one ester. In one embodiment, the chemical composition contains propanoic and isobutyric acids and at least one ester. In another embodiment, the two organic acids are propanoic and isobutyric acids and at least one ester is isoamyl butyrate. In another embodiment, the two organic acids are propanoic and isobutyric acids and at least one ester is isoamyl hexanoate. In another embodiment, the two organic acids are propanoic and isobutyric acids and at least one ester is isoamyl acetate.

As provided herein, the chemical composition may further comprise at least one ester. Consistent with this provision, that at least one ester may be any ester listed in Table 1 or elsewhere in this document. In some embodiments, the ester may have from 3 to 10 carbon atoms and any esters or combinations thereof may comprise at least 20% of the mixture. In one embodiment, the ester is an isoamyl ester. As provided herein, embodiments of the present invention can alternatively be designed using a whole family of isoamyl esters of various acids, ranging from C-6 (hexanoate) to C-12 (laurate), as well as various isoamyl alcohol esters with aromatic residues. acids such as esters of cinnamic acid, benzoate and phenylacetate. In one embodiment, the ester is isoamyl hexanoate. As used herein, the term "hexanoate" may mean a single type of hexanoate or may contain a mixture of the acid form of the hexanoate, including the branched forms. In another embodiment of the invention, the ester is isoamyl formate. In another embodiment of the invention, the ester is isoamyl butyrate. In another embodiment, the ester is isoamyl acetate. In one embodiment, the ester is isoamyl acetate. In one embodiment, the ester is selected from the group consisting of allyl acetate, n-decyl acetate, isoamyl acetate, phenethyl acetate. In one embodiment, the ester is strawberry aldehyde (ethyl 3-methyl-3-phenyl-ethylene oxide-2-carboxylate and an organic ester). In some embodiments of the invention, at least one ester can be any ester containing an acidic residue with one carbon. In one embodiment of the invention, at least one ester is isoamyl formate. In some embodiments of the invention, other compounds may be added to the composition as an ester. For example, the ester of octanoic acid and isoamyl alcohol is as active as the ester of lauric acid. Accordingly, the compositions of the present invention may contain the full range of isoamyl esters containing acid residues with 6-12 carbons. In one embodiment of the invention, the ester is an octanoic acid ester of isoamyl alcohol. In another embodiment of the invention, the ester is an ester of lauric acid and isoamyl alcohol. In some embodiments of the invention, benzene components such as benzoic acid ester, cinnamic acid ester and salicylic acid ester can be used. In one embodiment of the invention, the chemical composition contains propanoic acid and at least one ester containing an acidic residue with 6-12 carbon atoms.

In some embodiments of the invention, the compositions may contain mixtures of at least one organic acid and at least one ester in any proportions. In one embodiment of the invention, the ratio of at least one organic acid and at least one ester is about 6-7 to 2-3. In preferred embodiments of the invention, the ratio of at least one organic acid and at least one ester is about 7:2. In other embodiments of the invention, the compositions in accordance with the present invention may contain mixtures of two organic acids and at least one ester in any proportions. In one embodiment, the ratio of first organic acid: second organic acid: at least one ester is about 3.5:3.5:2 v/v/v. In another embodiment of the invention, the mixture of first organic acid: second organic acid: at least one ester is about 7 parts of two acids and 2 parts of the selected ester. In one embodiment of the invention, the chemical composition contains propanoic acid and at least one ester containing an acidic residue with 6-12 carbon atoms, and the chemical composition has a propanoic acid:ester ratio of about 7:2 v/v.

As provided herein, the present invention may include any chemical compositions with the addition of at least one endophytic fungus. The present invention is not limited to any particular fungus, however, an endophytic fungus is preferred, and fungi from the genus Fusarium are more preferred. The most preferred endophytic fungus is Fusarium subglutinans . In another embodiment, the endophytic fungus belongs to the genus Gloeosporium . As provided herein, the fungus may be contained in any composition by the addition of inoculated barley, or any other suitable carrier for the fungus, obvious to those skilled in the art.

In one embodiment, the chemical composition comprises organic acids and at least one ester and at least one fungus, wherein the two organic acids and at least one ester kill or reduce the growth of bacteria in human and animal excrement, and at least one fungus increases the rate of decomposition of human and animal excrement. In another embodiment of the invention, the composition contains propanoic acid, isobutyric acid of at least one ester and at least one fungus. In some embodiments of the invention, the compositions may further contain cineole, valencene, salts, or any other additive, filler, or other component necessary to create a composition having the desired characteristics.

In some embodiments of the invention, supplementing the culture of the fungus with additional compounds can increase the inhibitory properties of the fungus to a greater extent than any substances produced by the fungus or compound alone. Such activity is recognized as synergy. Thus, the present invention also provides chemical compositions that contain at least one fungus and at least one synergist. As used herein, the term "synergist" refers to any chemical compound which, when formulated with another compound, exhibits a higher bacterial inhibitory activity than that observed for each component formulated alone. Without being limited to the example, when the synergist is combined with the culture of the fungus, shows an increase in antimicrobial activity compared to the gas phase of the fungus or the synergist alone.

In some embodiments, the chemical compositions of the present invention may be used in combination with carriers such as zeolite or bentonite as a filler for cat litter, stable, barn, sheepfold, or small animal bed treatment. In such embodiments, the present invention inhibits faecal microorganisms such as E. coli and breaks down urea in the urine to ammonia. In another embodiment, a non-limiting carrier such as bentonite, zeolite, perlite, or other silica-based carriers may be added to the chemical compositions of the present invention in amounts that are effective in killing bacteria and reducing harmful and noxious odors.

In some embodiments, the compositions of the present invention may be mixed with soap or other dispersing solutions and may be used as an antimicrobial spray or on surfaces that are contaminated with bacteria or other microbes, such as surface treatments in hospitals, homes, food storage and preparation areas, including food processing plants, where fruits, meats, and more are prone to bacterial contamination and are a common problem.

In some embodiments of the invention, the composition in accordance with the present contains at least one chemical composition or preparation of the present invention. In one embodiment, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a drug of the invention and a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include Cremophor, or any other biological surfactant, as will be apparent to those skilled in the art. In one embodiment, the pharmaceutically acceptable carrier is a cremophor.

In some embodiments, the compositions of the present invention, and preferably C-3, can be used with a carrier (zeolite or bentonite or talc) to treat soil areas that are going to be used for seed production or for transplanting young seedlings, in order to reduce or eliminate infection.

In some embodiments of the invention, the compositions of the present invention may be mixed with detergents for cleaning carpet surfaces and bacterial decontamination of human and animal excrement or other biological contaminants of carpet surfaces.

In some embodiments, the compositions of the present invention may be used as an aerosol to clean fruits, vegetables, grains, and other agricultural products during planting, growth, harvesting, and/or during transport.

In some embodiments of the invention, the composition of the present invention may be applied or used, embedded or otherwise integrated into baby diapers, bandages or other devices in which bacterial decontamination is desired.

In some embodiments of the invention, the composition of the present invention may additionally contain a detergent, for the purpose of use as a soap for disinfecting the skin of humans and animals.

In some embodiments, the compositions of the present invention may be further formulated, incorporated, or otherwise integrated into candle wax to decontaminate the zone by evaporation during combustion.

In particular, the chemical compositions of the present invention exhibit high antibiotic activity against bacteria associated with human and animal excrement. These mixtures can be delivered specifically to the appropriate target site via inert carriers, such as, without limitation, bentonite, zeolite, perlite, or other silica-based carriers. In this case, in a specific place to use the carrier and antibiotic combination, for example, contained, without limitation, in the litter of pets and animals from the zoo. The mixture can be used in barns, bedding and animal habitats to reduce bacterial load and exposure to harmful gases.

It should be noted that the compositions of the present invention may contain any additional salts, excipients, food additives or impurities, and the like, so that the final composition is suitable for external use, ingestion, inhalation, or any other form of administration desired.

System 1

As described herein, the chemical composition may consist of two organic acids and at least one ester. For example, in one embodiment of the invention, the composition includes propanoic acid: isobutyric acid: isoamyl butyrate. In one embodiment, the ratio of propanoic acid: isobutyric acid: isoamyl butyrate is about 3.5:3.5:2 v/v/v. In another embodiment of the invention, the mixture of propanoic acid: isobutyric acid: isoamyl butyrate is about 7 parts of two acids and 2 parts of the selected ester. It should be noted that the chemical composition of system 1 is not limited to any particular ratio of such chemical components. In another embodiment of the invention, the chemical composition of system 1 contains only two organic acids and one ester. In this embodiment, the composition contains propanoic acid: isobutyric acid: isoamyl butyrate in the proportions indicated above. In some embodiments of the invention, the composition may additionally contain cineole, valencene, salts, or any other additives, fillers, or other components necessary to obtain a composition having the desired characteristics.

In another embodiment of the invention, a carrier, without limitation, such as bentonite, zeolite, perlite, or other silica-based carriers, may be added to the chemical composition of system 1 in amounts that are effective in killing bacteria and reducing harmful and noxious odors. This ratio is usually equal to 1 ml of system 1 to 224 g of carrier (volume/mass) or other appropriate ratios, without limitation, which are effective.

System 2

As provided herein, the present invention may include any chemical compositions of system 1 with the addition of at least one endophytic fungus. As demonstrated herein, endophytic fungi from the group, F. subglutinans and others, are most suitable for growing on human feces and degrading them. In addition, the fungus can grow on the combined liquid and solid waste when using the system 1 antimicrobial mixture, while bacteria and other microbes are killed, this mixture ensures maximum mold growth. In one embodiment, the fungus is Fusarium subglutinans . In one embodiment of the invention, the fungus is contained in system 2 by adding inoculated barley. In some embodiments of the invention, the composition may further contain cineole, valencene, salts, or any other additives, fillers, or other components necessary to obtain a composition having the desired characteristics.

In one embodiment of the invention, system 2 contains the chemical composition of system 1, for example, propanoic acid: isobutyric acid: isoamyl butyrate in a ratio of 3.5:3.5:2 v/v/v, or 7 parts of two acids and 2 parts of an ester, which is then added at a rate of 1/10 volume/weight (dry weight) into a mixture of a carrier material such as bentonite, perlite or zeolite, etc. It should be noted that the chemical composition of system 2 is not limited to any particular ratio of such chemical components. Barley is seeded with Fusarium subglutinans and also added to the composition. Then 10 g of this mixture is added to each container, such as a plastic bag, and used for the treatment and disposal of human excrement. This allows for rapid growth of Fusarium subglutinans as opposed to system 1 which does not. Other elements may also be added to the container, including liquid absorbent polymers in appropriate amounts, as will be appreciated by those skilled in the art,

System 3

As provided herein, the chemical composition of the present invention may contain at least one organic acid and at least one ester. In a preferred embodiment of the invention, at least one organic acid is propanoic acid. In one embodiment of the invention, at least one ester is isoamyl hexanoate or a mixture of isoamyl hexanoate. In a preferred embodiment of the invention, at least one ester is isoamyl hexanoate. In one embodiment, the chemical composition contains propanoic acid and isoamyl hexanoate. In some embodiments of the invention, the composition may further contain cineole, valencene, salts, or any other additives, fillers, or other components necessary to obtain a composition having the desired characteristics.

In one embodiment, the ratio of propanoic acid:isoamyl hexanoate is about 7:2 v/v. It should be noted that the chemical composition of system 3 is not limited to any particular ratio of such chemical components. In another embodiment of the invention, the chemical composition of system 3 contains a single organic acid component and a single ester component. In another embodiment of the invention, the chemical composition of system 3 contains a single organic acid component and a mixture of isoamyl hexanoate. In this embodiment, the composition contains propanoic acid: isoamyl hexanoate in the proportions indicated above. In another embodiment of the experiment, the chemical composition predominantly contains propanoic acid and isoamyl hexanoate in a propanoic acid: isoamyl hexanoate ratio of about 7:2 v/v.

In another embodiment, a non-limiting carrier such as bentonite, zeolite, perlite, or other silica-based carriers may be added to the system 3 chemical composition in amounts that are effective in killing bacteria and reducing harmful and noxious odors.

This ratio is typically 1.0 to 1.5 ml of system 3 per 224 g of carrier (v/w), or other appropriate ratios, without limitation, that are effective, such as 0.1 to 5 ml of system 3 per 224 g of carrier (v/w), or 0.5 to 2 ml of system 3 per 224 g of carrier (v/w).

System 4

As provided herein, the chemical composition of the present invention may contain at least one organic acid and at least one ester. In a preferred embodiment of the invention, at least one acid is propanoic acid. In one embodiment of the invention, at least one ester is isoamyl formate. In another embodiment of the invention, at least one ester may be an ester containing an acidic residue with one carbon atom. In one embodiment of the invention, the chemical composition contains propanoic acid and isoamyl formate. In some embodiments of the invention, the composition may additionally contain cineole, valencene, salts, or any other additives, fillers, or other components necessary to obtain a composition having the desired characteristics.

In one embodiment, the propanoic acid:isoamyl formate ratio is about 7:2 (v/v). It should be noted that the chemical composition of system 4 is not limited to any particular ratio of such chemical components. In another embodiment of the invention, the chemical composition of system 4 contains a single organic acid component and a single ester component. In such an embodiment of the invention, the composition contains propanoic acid: isoamyl formate in the proportions indicated above. In one embodiment, the chemical composition contains propanoic acid and isoamyl formate in a propanoic acid:isoamyl formate ratio of about 7:2 v/v.

As provided herein, the present invention may include any chemical compositions of system 4 with the addition of at least one endophytic fungus. As demonstrated herein, endophytic fungi from the group, F. subglutinans and others, are most suitable for growing on human feces and degrading them. In addition, the fungus can grow on the combined liquid and solid waste when using the system 4 antimicrobial mixture, while bacteria and other microbes are killed, this mixture ensures maximum mold growth. In one embodiment, the fungus is Fusarium subglutinans . In yet another embodiment of the invention, the present invention includes a chemical composition containing a mixture of propanoic acid and isoamyl formate in a ratio of 7 to 2, and optionally with the addition of Fusarium subglutinans . In this embodiment, the mixture of propanoic acid and isoamyl formate is suitable for killing selected microorganisms while not killing Fusarium spp., which further accelerates the disposal of the waste to which the composition is applied. In one embodiment of the invention, the fungus is contained in system 4 by adding inoculated barley.

In one embodiment of the invention, system 4 contains propanoic acid: isoamyl formate in a ratio of 7 to 2 v/v, which is then added to a carrier such as bentonite, perlite or zeolite, etc., in a ratio of 1/10 v/w (dry weight ). Barley is seeded with Fusarium subglutinans and also added to the composition. This mixture is then added to each container, such as a plastic bag, and used to treat and dispose of human feces. This ensures the rapid growth of Fusarium subglutinans . Other elements may also be added to the container, including liquid absorbent polymers in appropriate amounts, as will be understood by those skilled in the art,

System 5

As described herein, the chemical composition may contain two organic acids and at least one ester. For example, in one embodiment of the invention, the composition contains propanoic acid: isobutyric acid: isoamyl hexanoate. In one embodiment, the ratio of propanoic acid: isobutyric acid: isoamyl hexanoate is about 3.5:3.5:2 v/v/v. In another embodiment, the mixture of propanoic acid: isobutyric acid: isoamyl butyrate is about 7 parts of two acids and 2 parts of the selected ester. It should be noted that the chemical composition of system 5 is not limited to any particular ratio of such chemical components. In another embodiment of the invention, the chemical composition of system 5 contains two organic acids and one ester. In this embodiment, the composition contains propanoic acid: isobutyric acid: isoamyl hexanoate in the proportions indicated above. In some embodiments of the invention, the composition may further contain cineole, valencene, salts, or any other additives, fillers, or other components necessary to obtain a composition having the desired characteristics.

In another embodiment, a carrier, such as, but not limited to, bentonite, zeolite, perlite, or other silica-based carriers, may be added to the chemical composition of system 5 in amounts that are effective in killing bacteria and reducing harmful and noxious odors.

System X

As provided herein, the present invention may include any of the chemical compositions of systems 1-5 in combination with at least one salt, excipient, dietary supplement, or admixture. In a preferred embodiment of the invention, the chemical composition is system 3. As demonstrated herein, a chemical composition containing system 3 and at least one nutritional supplement and at least one salt is used to treat diseases and disorders associated with microbial infection. Examples of nutritional supplements include, but are not limited to, sugars such as glucose, sucrose, or fructose; amino acids such as glycine; and protein sources such as whey protein. Any protein source can be used, as will be understood by those skilled in the art. Non-limiting examples of salts include potassium chloride, sodium chloride, magnesium sulfate, potassium monophosphate, potassium sulfate, and magnesium acetate. Salts may be used in the compositions of the present invention as they enhance the electrolyte balance in the subject. Any amount of salt can be used in the compositions of the present invention. In a preferred embodiment, the amount of salt is greater than 0%. System 3 inhibits and kills pathogenic bacteria. In one embodiment of the invention, system X contains the chemical composition of system 3, glucose, whey protein, potassium chloride, magnesium sulfate, and sodium chloride. In another embodiment of the invention, system X contains the chemical composition of system 3, glucose, glycine, potassium chloride, sodium chloride and magnesium acetate. In another embodiment of the invention, system X contains the chemical composition of system 3, glucose, glycine, potassium chloride, sodium chloride, magnesium acetate and potassium monophosphate. It should be noted that the chemical composition of system X is not limited to any particular ratio of such chemical components. In one embodiment of the invention, the amount of organic acids is about 100% and the amount of esters is 0%. In another embodiment, the amount of organic acids is about 99% and the amount of esters is about 1%. In another embodiment, the amount of organic acids is about 1% and the amount of esters is about 99%.

In some embodiments, System X is formulated using one or more pharmaceutically acceptable excipients or carriers. Examples of pharmaceutically acceptable carriers include Cremophor, or any other biological surfactant as understood by those skilled in the art. In one embodiment, the pharmaceutically acceptable carrier is a cremophor. In one embodiment of the invention, system X contains the chemical composition of system 3 and cremophor.

In some embodiments, systems 2 and 4 can be used with or without a carrier to treat animal waste (including human feces) in the presence of Fusarium subglutinans . In such embodiments, the present invention inhibits and kills bacteria while allowing the growth of Fusarium subglutinans , which ultimately results in the breakdown or breakdown of solid material in human feces.

In some embodiments of the invention, the composition of the present invention is used to fumigate seeds contaminated with microorganisms.

In some embodiments of the invention, the composition of the present invention is administered in gaseous form without water or any additional carriers.

Ways

Decontamination of human excreta is only one problem associated with the waste treatment process. An additional problem considered within the framework of the present invention is the need for immediate degradation of organic matter in solid and liquid wastes. Biology and biochemistry take place when solid and liquid wastes form a complex. It turns out that urea in the urine is immediately exposed to the enzyme urease, found in most bacteria associated with solid waste, which is accompanied by the production of gaseous ammonia. The gas is harmful in itself and has a terrible smell. It is also fatal to most fungi as it causes the pH to rise. Thus, if one wants to induce waste degradation, it is necessary to stop ammonia production, which is desirable for fungal growth and ammonia remediation in the environment. Systems 1-4 each inhibit the growth of bacteria and their production of ammonia, causing the death of bacteria, and further systems 2 and 4 ensure the growth of Fusarium subglutinans, which then decomposes the waste. Thus, systems 1 and 3 are most suitable for treating animal bedding, waste, etc., reducing the amount of ammonia.

The discovery of a suitable microorganism that leads to the rapid decomposition of human and animal excrement began with bacteria living inside plants (namely, endophytes) - which is a suitable place to look. Endophytes are microorganisms that are the first to take part in the decomposition of a plant when it dies either by natural death or by external causes. They have a set of enzymes that breaks down the cellulose, lignin and hemicelluloses found in plant materials. These are the same complex organics found in human solid waste; therefore, in order to solve the problem to which the present application relates, namely the degradation of human and animal excrement, a number of endophytic microorganisms were found and examined for the ability of microorganisms to grow on both solid and liquid human excrement. In order for a microorganism to degrade waste, it must either be insensitive to ammonia, or ammonia must be removed from the equation. Thus, with the help of systems 2 and/or 4, which provide the growth of Fusarium spp. and eliminate ammonia production, it is possible to develop a useful and logical way to treat liquid and solid wastes.

In one aspect, the present invention includes a method for treating human and animal feces. In one embodiment of the invention, the method involves the interaction of human and animal excrement with a composition in accordance with the present invention, characterized in that the composition kills or reduces the growth of bacteria in human or animal excrement. In one embodiment of the invention, the composition contains a chemical composition in accordance with the present invention. In one embodiment of the invention, the chemical composition additionally contains at least one fungus. In another embodiment of the invention, at least one fungus increases the rate of decomposition of human and animal excrement.

In another aspect, the present invention includes a method for eliminating or reducing microbial growth at a treatment site. In one embodiment of the invention, the method includes contacting a composition of the present invention with a treatment site, characterized in that the composition kills or reduces the growth of bacteria in human and animal excrement. In one embodiment of the invention, the composition contains the chemical composition of the present invention. In one embodiment of the invention, the chemical composition further comprises at least one fungus.

In another aspect, the present invention includes a method for eliminating or reducing odor generation at a treatment site. In one embodiment of the invention, the method includes contacting a composition in accordance with the present invention with a treatment site, wherein the composition eliminates or reduces the formation of odor in human or animal excrement.

In another aspect, the present invention includes a method for eliminating or reducing the amount of ammonia at a treatment site. In one embodiment of the invention, the method includes interacting a composition in accordance with the present invention with a treatment site, and the composition eliminates or reduces the amount of ammonia in human or animal excrement.

In another aspect, the present invention includes a method for fumigating seeds contaminated with microorganisms. In one embodiment, the method includes contacting the seed with a composition of the present invention, wherein the composition reduces or eliminates the growth of microorganisms on the seed, and in some embodiments, reduces or eliminates the growth of microorganisms on the seed without substantially interrupting germination.

In some embodiments of the invention, the composition of the present invention can be used in hospitals for the treatment of human excrement in combination with a carrier placed in a medical vessel to stop contamination of the area with fecal bacteria. In some embodiments of the invention, the composition of the present invention can be used as an antiseptic for the treatment of cuts and wounds, superficial infections in animals and humans. For example, the present invention can be used to treat bacterial and viral intestinal infections in humans and animals. It should be noted that all the ingredients of system 1-4 belong to the list of substances recognized as completely harmless, and thus safe. In particular, 10 ml of C-3 was consumed by a person without side effects. Compositions and compositions according to the present invention can also be used for treating or disinfecting the surfaces of inanimate or inanimate objects, or spraying, or topically applied to all types of plants, such as agricultural fruits, vegetables, cereals, and the like, or can be applied topically, by enteral or inhalation route in any type of animal, such as livestock, or in humans.

In one aspect, the present invention includes a method for preserving fruits. In one embodiment, the method comprises administering to the fetus an effective amount of a composition according to the present invention. In one embodiment, the fruits are raspberries or grapes.

In some embodiments of the invention, the composition of the present invention, preferably C-3, can be used to disinfect corn used for fermentation into alcohol.

Mastitis is an infection of the tissues of the cow's udder. Almost any bacterial or fungal organism that can invade tissue and cause infection can cause mastitis. It represents one of the most important problems in dairy production. Most cases of mastitis infection are caused by various types of streptococci, staphylococci, and gram-negative rods, especially lactose-fermenting organisms of intestinal origin, commonly referred to as coliform bacteria, and these include organisms such as E. coli and Staphylococcus aureus. From an epidemiological point of view, the source of infection can be considered infectious or exogenous, and cows are in constant danger of being infected with these agents.

In addition to Mycoplasma spp , which can be airborne from cow to cow and invade the udder to cause bacteremia, infectious pathogens are spread during milking by the hands of the milkmaid or by the milking machine line. The main bacterial species that use this mode of transmission include Staphylococcus aureus , Streptococcus agalactiae , and Corynebacterium bovis . Most other species are opportunistic to cows in the environment, although some other streptococci and staphylococci may also be infectious.

Intramammary infections are often referred to as subclinical or clinical mastitis. Subclinical mastitis is the presence of infection without visible signs of local inflammation or systemic involvement. Although transient episodes of abnormal milk or udder inflammation may occur, these infections are most often asymptomatic and, if the infection persists for at least 2 months, is termed chronic. Once established, many of these infections persist throughout the lactation or life of the cow. Detections are best made by testing the milk for somatic cell counts (predominantly neutrophils) or using the California Mastitis Test or automated methods to improve dairy herd organization. Somatic cells are positively correlated with the presence of infection. Although variable (especially if the assay was performed once), cows with a somatic cell count ≥280,000 cells/mL (≥ linear score 5) have a >80% chance of infection. Similarly, the more somatic cells in the bulk milk tank, the higher the prevalence of infection in the herd. Pathogens must be identified by bacterial culture of milk.

Clinical mastitis is an inflammatory response to infection resulting in apparently abnormal milk (eg, color, fibrin clots). As the degree of inflammation increases, changes in the udder (swelling, heat, pain, redness) may also appear. Clinical cases that include only local signs are called mild or moderate. If the inflammatory process includes systemic involvement (fever, anorexia, shock), then the case is called severe. If the onset of the disease is very rapid, as often happens in severe clinical cases, then this is called an acute case of severe mastitis. More severely affected cows tend to have more serous discharge in the affected udder quarter.

Although any number of quarters may be infected with subclinical mastitis at the same time, typically only one quarter at a time will show clinical mastitis. However, it is not uncommon for clinical episodes caused by mycoplasma to affect multiple quarters. Gangrenous mastitis may become more severe during periods of immunosuppression (eg, during childbirth), particularly in subclinical, chronic Staphylococcus aureus infections. In subclinical mastitis, culture of milk samples collected from affected quarters is the only reliable way to determine the etiology of clinical cases.

All dairy herds have cows with subclinical mastitis; however, the prevalence of infected cows ranges from 15 to 75% and udder quarters from 5 to 40%. Various pathogens can cause chronic infection, but only in some cases will there be clinical signs of mastitis. The main goal in most programs to control subclinical mastitis is to reduce the prevalence of infectious pathogens Streptococcus agalactiae and Staphylococcus aureus, as well as other gram-positive cocci, especially Streptococcus dysgalactiae (which can also be contagious or environmental) , Streptococcus uberis, enterococci and numerous other coagulase-negative staphylococci , including S hyicus , S epidermidis , S xylosus , and S intermedius.

For infectious pathogens, adult cattle that are being milked are most at risk of infection, either during lactation or during the dry period. The main receptacle of infection are the mammary glands; infection occurs during milking by the hands of a milkmaid or by milking equipment acting as a mechanical carrier. For the first time, pregnant heifers were reported to be infected with staphylococci and streptococci before calving, although the prevalence of infection varies greatly among herds and geographic regions. The cause of dermatitis of the bovine mammary glands is the small cow's fly ( Haematobia irritans ), which can be a site of accumulation of Staphylococcus aureus and is associated with an increased risk of infection of heifers, especially in warm climates.

Commonly used treatments include the use of antibiotics, which pose a threat to the milk received from the animal, as the antibiotics will be reflected in the udder. Milk cannot be used for at least 3 days after antibiotic administration. The use of immunization is not possible because there are a large number of potential pathogens involved in mastitis. The main recommendations for improving sanitation are: increasing cleanliness in the milking parlour, and in areas frequented by animals. Currently, no available approaches have been found to be effective and safe for the treatment of mastitis.

In one aspect, the present invention includes a method of treating an animal having a disease or disorder associated with a microbial infection. In one embodiment, the method comprises administering to an animal an effective amount of a composition according to the present invention. In another embodiment of the invention, the method includes administering to the animal an effective amount of a composition containing an organic acid. Such diseases and disorders may include, without limitation, such as diarrhea, food poisoning, or indigestion, or intramammary infection such as subclinical or clinical mastitis. It should also be understood that the compositions and compositions of the present invention are not limited to the treatment of any particular type of object. Under this provision, the subject may be any animal, preferably a mammal, more preferably livestock such as cattle, sheep, or pigs, or even a human. In one embodiment of the invention, the animal is cattle, pigs and sheep. In another embodiment of the invention, the animal is a human.

In another aspect, the present invention includes a method of treating cows having diarrhea. In one embodiment, the method comprises administering to the cow an effective amount of a composition according to the present invention.

In another aspect, the present invention includes a method of treating pigs having diarrhoea. In one embodiment, the method comprises administering to the pig an effective amount of a composition according to the present invention.

In another aspect, the present invention includes a method of treating cows with mastitis. In one embodiment, the method comprises administering to the cow an effective amount of a composition according to the present invention.

In another aspect, the present invention includes a method of treating sheep that have mastitis. In one embodiment, the method comprises administering to the sheep an effective amount of a composition according to the present invention.

In another aspect, the present invention includes a method of treating a human having a gastrointestinal disease. In one embodiment, the method comprises administering to a human an effective amount of a composition according to the present invention. In one embodiment, the gastrointestinal disease is food poisoning or gastroenteritis.

Complex therapy

The compositions of the present invention are intended for possible use in combination with one or more additional substances. Without being limited to examples, compositions of the invention may be used in combination with one or more therapeutic agents (salts, solvates or prodrugs). Non-limiting examples of therapeutic agents include antibiotics such as Baytril, sulfonamides, Nuflor, Tilan 40-50, Exid, Oxytetracycline LA, Draxin, and tetracycline, vaccines such as Enforce 3, multivitamins, probiotics, and toxin absorbents such as Toxiban, or other therapeutic agents such as Suprio.

In another embodiment, the compositions of the present invention may be used in combination with a detergent. In one embodiment of the invention, the detergent acts as a solvent for the composition to remove unwanted bacterial contamination from infected areas of the subject, and any other possible sources of infection, such as bedding, tools, or places where the subject lives. As a non-limiting example, the compositions of the present invention can be used in the treatment of udders, bedding of cattle, which are the main sources of pathogen contamination, as well as tools used in the milking process, which have been identified as potential sources of infection, such as contaminated solutions for udder teat treatments, intramammary injections, water hoses used in preparing udders for milking, pools or potholes for shoveling dirt, skin lesions, teat injury, and flies. Non-limiting examples of detergents include Sucragel CF, Chemoxide CAW, BioSoft D40, Lathanol LAL, BioTerge AS-40, Nacconol 90G, and potassium cocoate.

Pharmaceutical compositions and methods of treatment

Successful administration of a composition within the scope of the present invention may be accomplished in many different ways, using methods generally accepted in the art. Therapeutic and prophylactic methods according to the present invention thus encompass the use of pharmaceutical compositions which comprise the compositions used in the methods of practice according to the present invention. Pharmaceutical compositions that are used in the practice of the invention may be administered at doses ranging from 1 mg/kg/day to 100 mg/kg/day.

The relative amount of active substance, with a pharmaceutically acceptable carrier, and any additional substance in a pharmaceutical composition in accordance with the present invention varies depending on the individual, size, condition of the subject and further depending on the method by which the composition is administered. For example, the composition may contain from 0.1% to 100% (w/w) active agent.

While the description of the pharmaceutical compositions provided herein principally relates to pharmaceutical compositions that are suitable for administration to humans, one of ordinary skill in the art will appreciate that such compositions are generally suitable for administration to animals of all kinds. Modification of pharmaceutical compositions suitable for administration to humans to render the composition suitable for administration to various animals is well understood and typically such modification can be designed and performed by a skilled veterinarian/pharmacologist with little more than routine experimentation. Subjects to whom the pharmaceutical compositions of the present invention are administered include, but are not limited to, humans and other primates, mammals, including commercial mammals such as non-human primates, cattle, pigs, horses , sheep, cats and dogs.

Typically, doses that can be administered by the method of the present invention to an animal, preferably a human, are in the range of 0.5 μg to about 50 mg per kilogram of animal body weight. While the exact dose will vary depending on any number of factors, including, but not limited to, the species of animal and disease being treated, the age of the animal, and the route of administration, the dosage of the composition preferably ranges from about 1 μg to 10 mg per kilogram of the animal's body weight. More preferably, the dosage ranges from about 3 µg to 1 mg per second.

Pharmaceutical compositions used in the methods of the present invention may be formulated, packaged, or sold in compositions suitable for oral, parenteral, topical, buccal, or other routes of administration. Other envisaged compositions include putative nanoparticles, liposome preparations, anti-caking agents, and immunologically active compositions.

The pharmaceutical compositions described herein may be prepared by any of the conventional methods, or will be developed later in pharmacology. In general, such preparation methods include the step of bringing the active agent into association with a pharmaceutically acceptable carrier or one or more other additional ingredients, and then, if necessary or appropriate, filling and packaging the product in the desired single or multi-dose container. The pharmaceutical composition in accordance with the present invention may be prepared, packaged or sold by weight, in the form of a single or multiple unit doses. As used herein, a "single dose" is a discrete amount of a pharmaceutical composition containing a specific amount of an active substance. The amount of active agent will typically be the dose of active agent to be administered to the subject, or a convenient fraction of such dose, such as, for example, half or a third of such dose.

In one embodiment of the invention, the composition in accordance with the present invention is prepared using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a composition of the present invention and a pharmaceutically acceptable carrier thereof. Pharmaceutically acceptable carriers that are used include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable saline solutions such as phosphates, and organic acid salts. Examples of these and other pharmaceutically acceptable carriers are described in Remington Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures, and vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. Prevention of the influence of microorganisms can be achieved with various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases it will be preferred to include isotonic agents, eg sugars, sodium chloride, or polyols such as mannitol and sorbitol, in the composition. Prolongation of the absorption of injectable compositions can be accomplished by including in the composition an agent that delays absorption, such as aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not just DMSO.

The compositions may be used in admixture with conventional carriers, ie. pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable route of administration known in the art. Pharmaceutical preparations may be sterilized and preferably mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect the osmotic pressure in buffer solutions, coloring agents, flavoring and/or aromatic substances and the like. . They may also be combined, if desired, with other active agents, such as other analgesics.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; tearing and loosening substances; binding agents; lubricating agents; sweeteners; flavoring agents; dyes; preservatives; physiologically degradable compositions such as gelatin; aqueous solvents and solvents; oil solvents and solvents; suspending agents; dispersing or wetting agents; emulsifiers, sedatives; buffer; salt; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizing agents; and their pharmaceutically acceptable polymeric or hydrophobic substances. Other "additional ingredients" that may be contained in pharmaceutical compositions in accordance with the present invention are known in the art and are described, for example, in Genaro, ed. (1985, Remington Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

The pharmaceutical composition in accordance with the present invention may contain a preservative from about 0.005% to 2.0% of the total weight of the composition. A preservative is used to prevent spoilage in case of exposure to environmental contaminants. Examples of preservatives used in accordance with the present invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidourea, and combinations thereof. The most preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

The pharmaceutical composition preferably includes an antioxidant and a chelating agent that inhibits degradation of the drug. Preferred antioxidants for some compositions are butylated hydroxytoluene, butylated hydroxyanisole, alpha-tocopherol, and ascorbic acid in a preferred range of about 0.01% to 0.3%, and a more preferred range for butylated hydroxytoluene is 0.03% to 0.1% by weight. from the total weight of the composition. Preferably, the chelating agent is present in an amount of 0.01% to 0.5% by weight of the total weight of the composition. The most preferred chelating agents include ethylenediaminetetraacetate (e.g., disodium edetate) and citric acid in the weight range from about 0.01% to 0.20%, and in the more preferred range from 0.02% to 0.10%, by weight of the total weight. compositions. The chelating agent used to chelate the metal ions in the formulation can adversely affect the shelf life of the formulation. While butylhydroxytoluene and disodium edetate are the most preferred antioxidant and chelating agent, respectively, for some compositions, other antioxidants and chelating agents may be most suitable and equivalent, and therefore should be known to those skilled in the art.

Liquid suspensions may be prepared using conventional methods for suspension of the active ingredient in an aqueous or oily vehicle. Aqueous solvents include, for example, water, saline and isotonic saline. Oil solvents include, for example, almond oil, fatty esters, ethyl alcohol, vegetable oils such as peanut, olive, sesame, coconut or rectified vegetable oils and mineral oils such as liquid paraffin. Aqueous suspensions may additionally contain one or more "additional ingredients", including, but not limited to, suspending agents, dispersing or wetting agents, emulsifiers, demulcents, preservatives, buffers, salts, flavors, colors, and sweeteners. Oil suspensions may also contain a thickener. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum arabic, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, stearoxyhydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides such as lecithin, condensates of alkylene oxide with a long chain fatty acid aliphatic alcohol, with a partial ester derived from a fatty acid and hexitol or a partial ester derived from a fatty acid. and hexitan anhydride (eg, polyoxyethylene stearate, heptadecaethylene oxycetanol, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin and gum arabic. Known preservatives include, but are not limited to, methyl, ethyl or n-propyl parahydroxybenzoate, ascorbic acid, and sorbic acid. Known sweeteners include, for example, glycerin, propylene glycol, sorbitol, sucrose, and saccharin. Known thickeners for oil suspensions include, for example, beeswax, paraffin wax and cetyl alcohol.

Liquid solutions of the active substance in aqueous or oily solvents can be prepared in much the same way as liquid suspensions, the main difference being that the active substance is dissolved rather than suspended in a solvent. As used herein, an "oily" liquid is one that contains a liquid hydrocarbon molecule and that exhibits less polar properties than water. Liquid solutions of a pharmaceutical composition according to the present invention may contain each of the components described in connection with liquid suspensions, and it should be noted that suspending agents do not necessarily help dissolve the active substance in the solvent. Aqueous solvents include, for example, water, saline and isotonic saline. Oil solvents include, for example, almond oil, fatty esters, ethyl alcohol, vegetable oils such as peanut, olive, sesame, coconut or rectified vegetable oils and mineral oils such as liquid paraffin.

Powdered and granular compositions of the pharmaceutical composition in accordance with the present invention can be obtained using conventional methods. Such compositions may be administered directly to the subject, for example, in the form of tablets, capsules, or prepared in an aqueous or oily suspension or solution with an aqueous or oily solvent added thereto. Each of these compositions may additionally contain one or more dispersing or wetting agents, suspending agents and preservatives. Additional excipients such as fillers and sweeteners, flavors or colors may also be included in these compositions.

The pharmaceutical composition according to the present invention may also be formulated, packaged or sold as an oil-in-water emulsion or as a water-fat emulsion. The oil phase may be a vegetable oil such as olive or peanut oil, a mineral oil such as liquid paraffin, or combinations thereof. Such compositions may additionally contain one or more emulsifying agents such as natural gums such as gum arabic or gum tragacanth, naturally occurring phosphatides such as soy or lecithin, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides, such as sorbitan monooleate, and the condensation products of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweeteners or flavors.

Methods for impregnating or applying a chemical composition to a material, as known in the art, include, but are not limited to, methods for applying or fixing a chemical composition to a surface, methods for incorporating the chemical composition into the structure of the material during material synthesis (i.e., for example, with physiologically degradable material), and methods for absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

Controlling or slowing down the release of a composition from a preparation in accordance with the present invention can be done using conventional technology, in addition to the invention set forth in this description. In some cases, the dosage forms to be used may be able to release one or more active substances in a delayed or controlled manner using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or or their combinations to provide the desired release profile in various proportions. Suitable controlled release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the compositions of the present invention.

The controlled release of the active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water or other physiological conditions or compounds. The term "controlled release of a component" in the context of the present invention is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, nanoparticles, or microspheres, or combinations thereof, which facilitate controlled release. active ingredient.

Introduction/Dosing

The mode of administration may affect how much of the composition is effective. Therapeutic compositions may be administered to a subject either before or after the disease has been identified. In addition, multiple separate doses, such as staggered or daily or sequential doses, or bolus administration of a substance. In addition, doses of therapeutic compositions may be proportionally increased or decreased as indicated by acute therapeutic or prophylactic situations.

Administration of the compositions of the present invention to a subject, preferably a mammal, more preferably a human, may be carried out using conventional methods, at doses and for a period of time effective to prevent or treat the disease. The effective amount of the therapeutic composition required to achieve a therapeutic effect may vary depending on factors such as the potency of the particular composition used; time of administration; the rate of removal of the composition; duration of treatment; other drugs, compositions or substances used in combination with the composition; the course of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical field. The dosage regimen may be varied to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the therapeutic situation. A non-limiting example of an effective dosage range for a therapeutic composition of the present invention is about 1 to 5000 mg/kg body weight/day. One skilled in the art will be able to study the relevant factors and make a determination of the effective amount of the therapeutic composition without undue experimentation.

The composition may be administered to the animal at a frequency of several times a day, or may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months, or even once a year or less. Dose frequency will be apparent to those skilled in the art and will depend on any number of factors such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, and so on. Compositions from the pharmaceutical compositions described herein can be prepared by any of the conventional methods or methods that will be developed later in pharmacology. In general, such preparations include the step of bringing the active ingredient into association with the carrier or one or more other adjuncts and then, if necessary, filling and packaging the product in the desired single or multi-dose containers.

Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition and route of administration, while not being toxic to the subject.

A physician, such as a physician or veterinarian, or one of ordinary skill in the art, can easily determine and prescribe the effective amount of the pharmaceutical composition that is required. For example, a physician or veterinarian may dose the compositions of the present invention, use the pharmaceutical composition at levels below those required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

In a specific embodiment of the invention, it is most advantageous to design compositions in the form of a dispenser for ease of administration and dose uniformity. Dosage form as used herein refers to physically discrete units suitable as unit doses for subjects to be treated; each block containing a predetermined amount of therapeutic composition calculated to produce the desired therapeutic effect, in association with the desired carrier. The dosage form in accordance with the present invention is dictated by and directly dependent on (a) the unique characteristics of the therapeutic composition and the particular therapeutic effect to be achieved, and (b) the limitations in the art inherent in the mixture/composition in such a therapeutic composition for the treatment of disease in subject.

In one embodiment of the invention, compositions in accordance with the present invention are administered to a subject at doses that range from one to five times a day or more. In another embodiment, the compositions of the present invention are administered to a subject at a dosage range that includes, but is not limited to, once a day, every two days, every three days or once a week, and once every two weeks. It will be apparent to one of ordinary skill in the art that the frequency of application of various combinations of compositions in accordance with the present invention will vary from subject to subject depending on many factors, including, but not limited to, age, disease or disorder being treated, gender, general health status and other factors. Thus, the invention is not to be construed as limiting any particular dosage and precise dosage regimen, and the composition to be applied to any subject will be determined by the attending physician, taking into account all other factors about the subject.

Compositions according to the present invention for administration range from about 0.1 mg to 1000 mg, about 0.2 mg to 950 mg, about 0.4 mg to 900 mg, about 1 mg to 850 mg, about 5 mg to 750 mg, about 20 mg to 700 mg, about 30 mg to 600 mg, about 50 mg to 500 mg, about 75 mg to 400 mg, about 100 mg to 300 mg, about 120 mg to 250 mg, and with any integer or fractional step between them.

In some embodiments of the invention, the dose of the composition in accordance with the present invention is from about 1 mg to 2500 mg. In some embodiments of the invention, the dose of the composition in accordance with the present invention, used in the compositions described herein, is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3000 mg, or less than about 2000 mg, or less than about 1000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments of the invention, the dose of the second composition (i.e., the drug used to treat the same or a different disease, which is treated with compositions in accordance with the present invention), as described herein, is less than about 1000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any integer or fractional step in between.

In one embodiment of the invention, the present invention relates to the packaging of a pharmaceutical composition in a container containing a therapeutically effective amount of the composition in accordance with the present invention, alone or in combination with a second pharmaceutical agent; and instructions for using the composition to treat, prevent, or reduce one or more symptoms of a disease in a subject.

Mode of application

Methods for administering any of the compositions of the present invention include oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal (trans)urethral, vaginal (e.g., trans- and paravaginal), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, endobronchial, inhalation and topical.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, tablets, gel capsules, lozenges, dispersions, suspensions, solutions, syrups, granules, boluses, transdermal patches, gels, powders, granules, massecuites, lozenges, creams, pastes. , patches, lotions, discs, suppositories, liquids, sprays for nasal or oral use, dry powder or aerosol dosage forms for inhalation, compositions and preparations for intravesical administration, and the like. It should be understood that the formulations and compositions used in the present invention are not limited to the specific formulations and compositions described herein.

Oral administration

For oral administration, tablets, dragees, liquids, drops, suppositories or capsules, caplets and gelatin capsules are most suitable. Other formulations suitable for oral administration include, but are not limited to, powder or granular formulation, aqueous or oily suspension, aqueous or oily solution, paste, gel, toothpaste, mouthwash, coating and mouthrinse, or emulsion. . Compositions intended for oral use may be prepared according to any method known to those skilled in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutical excipients which are suitable for the manufacture tablets. Such excipients include, for example, an inert diluent such as lactose; grinder and baking powder, such as corn starch; binding agents such as starch; lubricating agents such as magnesium stearate.

Tablets may be uncoated, or they may be coated by known methods to achieve delayed disintegration in the subject's gastrointestinal tract, thereby providing sustained release and absorption of the active ingredient. For example, a substance such as glyceryl monostearate or glyceryl distearate can be used to coat tablets. Further, for example, tablets may be coated using the methods described in US Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to provide osmotically controlled release tablets. Tablets may additionally contain a flavoring agent, a flavoring agent, a coloring agent, a preservative, or any combination thereof, in order to provide a pharmaceutically original and palatable formulation.

Hard capsules containing the active substance can be prepared with physiologically degradable compositions such as gelatin. Such hard gelatin capsules consist of the active ingredient, and may further contain additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin.

Soft gelatin capsules containing the active substance can be prepared using physiologically degradable compositions such as gelatin. Such soft gelatin capsules which contain an active substance which can be mixed with water or an oily medium such as peanut oil, liquid paraffin or olive oil.

Compositions according to the present invention for oral use may be in the form of tablets or capsules prepared in a known manner with pharmaceutically acceptable excipients such as astringents; fillers; lubricants; baking powder; or wetting agents. If desired, tablets may be coated using suitable coating methods and materials such as OPADRY™ film coating systems from Colorcon, West Point, PA (e.g. OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400).

Liquid oral preparations may be in the form of solutions, syrups or suspensions. Liquid preparations may be prepared by known methods with pharmaceutically acceptable additives such as suspending agents (eg sorbitol syrup, methyl cellulose or edible hydrogenated fats); emulsifiers (eg lecithin or gum arabic); anhydrous solvents (eg almond oil, fatty ester or ethyl alcohol); and preservatives (eg methyl or propyl parahydroxybenzoate or sorbic acid). Liquid dosage forms of a pharmaceutical composition in accordance with the present invention suitable for oral administration may be prepared, packaged and sold either in liquid form or in the form of a dry product intended to be diluted with water or other suitable solvent before use.

A tablet containing the active substance may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets can be prepared by compressing in a suitable device the active substance in free-flowing form, such as a powder or granular preparation, optionally mixed with one or more of binders, lubricants, fillers, surfactants, and dispersants. Molded tablets can be made by molding in a suitable machine a mixture of the active ingredient and a pharmaceutically acceptable carrier and at least a sufficient amount of liquid to moisten the mixture. Pharmaceutically acceptable carriers useful in the manufacture of tablets include, but are not limited to, inert diluents, tearing and disintegrating agents, binders and lubricants. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, dicalcium phosphate, and sodium phosphate. Known tearing and disintegrating agents include, but are not limited to, cornstarch and alginic acid. Known binders include, but are not limited to, gelatin, gum arabic, pregelatinized cornstarch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Granulation methods for modifying starting powders or other active ingredient solids are well known in the pharmaceutical art. Powders, usually mixed with a binder into larger, permanent free-flowing agglomerates or granules, are referred to as "granular". For example, a "wet" granulation process using a solvent, typically characterized in that the powders are combined with a binder or wetted with water or an organic solvent under conditions resulting in the formation of a wet granulation mass, from which the solvent is then evaporated.

Melt granulation generally consists of using substances that are solid or semi-solid at room temperature (i.e. having a relatively low softening point and melting range) to aid in the granulation of powdered or other materials, essentially without the addition of water or other liquid solvents. Solids with low melting points, when heated to a temperature in the melting range, liquefy and act as a binder when the carrier is granulated. The liquefied solids themselves spread over the surface of the powdery materials with which they have bonded, and when cooled, form a solid granular mass in which the original substances are associated with each other. As a result of granulation, the melt may be fed into a tablet press or be encapsulated to prepare an oral dosage form. Melt granulation increases the dissolution rate and bioavailability of the active (i.e. drug) by forming a solid dispersion or thick solution.

US Patent No. 5169645 directly describes compressible granules containing wax and having improved flow properties. Granules are also obtained when waxes are mixed into the melt with certain additives that improve fluidity, subsequently cooling and granulating this mixture. In some embodiments, only the wax alone melts into the mixture of wax(s) and additive(s), and in other cases, the wax(s) and additive(s) will melt.

The present invention also includes a multilayer tablet comprising a layer providing a sustained release of one or more compositions in accordance with the present invention and an additional layer providing an immediate release of a drug for the treatment of a disease. Using a wax/mixture of pH sensitive polymers, a composition insoluble in gastric juice can be obtained, which contains the active substance, thereby providing its delayed release.

parenteral administration

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical disruption of the subject's tissues and administration of the pharmaceutical composition through a puncture into the tissues. Thus, parenteral administration includes, but is not limited to, administering a pharmaceutical composition by injecting the composition, by applying the composition through a surgical incision, by applying the composition through penetrating non-surgical wounds, and the like. In particular, parenteral administration includes, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular injection, intratumoral, and peritoneal dialysis techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration include the active substance in combination with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged or sold in unit dosage form, for example in ampoules or in multi-dose containers containing a preservative. Compositions for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained release or biodegradable formulations. Such preparations may additionally contain one or more additional ingredients, including, but not limited to, suspendresume, stabilizing or dispersing agents. In one embodiment, the parenteral composition comprises the active substance in dry form (ie, powder or granules) for reconstitution with a suitable vehicle (eg, sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition.

Pharmaceutical compositions may be prepared, packaged, or sold as sterile injectable aqueous or oily suspensions or solutions. This suspension or solution may be prepared in accordance with the prior art and may contain, in addition to the active ingredient, additional agents such as dispersing agents, wetting agents, or suspendida agents described herein. Such sterile injectable compositions may be prepared using a non-toxic parenterally acceptable diluent or solvent, for example in water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parenterally administered compositions that may be used include those containing the active substance in microcrystalline form, in liposomal formulations, or as a component of biodegradable polymer systems. Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic substances such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

Local application

The pharmaceutical composition according to the present invention may be formulated, packaged or sold in a formulation suitable for topical use. There are several advantages in delivering a composition, including drugs or other drugs, to the skin (dermal drug delivery) or to the body through the skin (transdermal drug delivery). Transdermal delivery of the composition offers an attractive alternative to injection and oral medications. Dermal delivery of a composition provides an efficient way to deliver the composition to the skin of a mammal, preferably a human, and provides a method of treating the skin, or otherwise providing the skin without the need to damage or compromise the integrity of the outer layer of the skin. In the present invention, dermal delivery by exposing the skin to the composition of the present invention provides the following benefits for the treatment of skin-related disorders or diseases.

A number of compounds, including some drugs, will effectively penetrate the skin simply because the molecules are small and potent at low doses of 0.1 mg to 15 mg/day (Kanikkannan et al., 2000, Curr. Med. Chem. 7:593-608). Many other compounds and drugs can only be delivered if there is an additional amplification system that "forces" them through the skin. Several methods of transdermal drug delivery include electroporation, sonophoresis, iontophoresis, penetration enhancers (cyclodextrins), and liposomes. Although the aforementioned methods are also included in the present invention for dermal delivery of compositions in accordance with the present invention, liposomes are the preferred dermal drug delivery method.

Compositions in accordance with the present invention may contain the active agent alone, in a form suitable for administration to a subject, or the composition may contain at least one active agent and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combinations thereof. The active ingredient may be present in the composition in the form of a physiologically acceptable ester or salt, for example in combination with a physiologically acceptable cation or anion, as is well known in the art. The compositions of the present invention should also be understood to cover pharmaceutical compositions used in the treatment of other conditions, disorders and diseases associated with the skin.

In one aspect, dermal drug delivery in accordance with the present invention is a composition containing at least one first compound that can facilitate dermal delivery of at least one second compound associated with or in close physical proximity to the composition containing first connection. As will be understood by those skilled in the art, ready for the description set forth herein, such delivery vehicles include, but are not limited to, liposomes, nanosomes, phospholipid non-liposome compositions (eg, selected cochleates), among others.

Compositions suitable for topical application include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, water-fat or oil-in-water emulsions such as creams, ointments or pastes, or solutions and suspensions. Topical preparations may, for example, contain from about 0.001% to about 90% (w/w) of the active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Compositions for topical application may additionally contain one or more additional ingredients described in this document.

In one aspect of the present invention, the dermal delivery system includes a liposome delivery system, and the present invention is not to be construed as limited to any particular liposome delivery system. Based on the description set forth herein, those skilled in the art will understand how the liposome delivery system used in the present invention is defined.

The present invention also encompasses the improvement of dermal and transdermal drug delivery by using absorption enhancers (also called sorption promoters or catalysts) that penetrate the skin to reversibly lower the resistance barrier. Many substances known in the art to enhance penetrating activity, including alkyl sulphide oxide (such as dimethyl sulfoxide, DMSO), azones (such as laurocapram), ketopirollidine (such as 2-pyrrolidone, 2P), alcohols, and alkanols (ethanol, or decanol ), glycols (eg, propylene glycol, PG, a common topical excipient), surfactants (also common in pharmaceuticals), and terpenes. Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, saturated carboxylic acid, dimethyl sulfoxide, polar lipids, or n-methyl-2-pyrrolidone.

In alternative embodiments, the topical pharmaceutical or cosmetic compositions may optionally be combined with other ingredients such as humectants, cosmetic adjuvants, antioxidants, chelating agents, surfactants, blowing agents, conditioners, humectants, wetting agents, emulsifiers, fragrances. , thickeners, buffering agents, preservatives, sunscreens, and the like. In another embodiment, penetration and absorption enhancers are included in the composition and are effective in improving the transdermal penetration of the active ingredients through the stratum corneum relative to the composition devoid of the penetration enhancer. Various penetration enhancers, including oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, saturated carboxylic acid, dimethyl sulfoxide, polar lipids, or n-methyl-2-pyrrolidone, are known to those skilled in the art.

In another aspect, the composition may further comprise a hydrotrope which acts to increase disorder in the structure of the stratum corneum, and thereby allows increased transport through the stratum corneum. Various hydrotropic substances such as isopropyl alcohol, propylene glycol, sodium xylene sulfonate are known to those skilled in the art. Compositions of this invention may also contain an active amount of retinoids (i.e., compounds that bind to any of the retinoid family receptors), including, for example, tretinoin, retinol, tretinoin and/or retinol esters, and the like.

Compositions in accordance with the present invention may contain a preservative from about 0.005% to 2.0% of the total weight of the composition. The preservative is used to prevent spoilage in case of repeated use by patients of the aqueous gel when it is exposed to environmental contaminants, such as air or the patient's skin, including contact with the fingers used to apply the compositions of the present invention, such as a therapeutic gel or cream.

Examples of preservatives used in accordance with the present invention include, but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidomyourea, and combinations thereof. The most preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

The composition preferably includes an antioxidant and a chelating agent that inhibits degradation of the composition when an aqueous gel composition is used in the present invention. Preferred antioxidants for certain compounds are butylated hydroxytoluene, butylated hydroxyanisole, alpha-tocopherol and ascorbic acid in a preferred range of about 0.01% to 5%, and a more preferred range for butylhydroxytoluene is 0.01% to 1% by weight of the total weight of the composition. Preferably, the chelating agent is present in an amount of 0.01% to 0.5% by weight based on the total weight of the composition. The most preferred chelating agents include ethylenediaminetetraacetate (e.g., disodium edetate) and citric acid in the weight range from about 0.01% to 0.20%, and in the more preferred range from 0.02% to 0.10%, by weight of the total weight. compositions. The chelating agent used to chelate the metal ions in the formulation can adversely affect the shelf life of the formulation. While butylhydroxytoluene and disodium edetate are the most preferred antioxidant and chelating agent, respectively, for some compositions, other antioxidants and chelating agents may be most suitable and equivalent, and therefore should be known to those skilled in the art.

Additional components may include, but are not limited to, water, oils (eg olive oil/PEG7), aloe oil, wax (eg jojoba wax), squalene, myristate (eg isopropyl myristate), triglycerides (eg caprylic triglyceride ), Solulan 98, cocoa butter, shea butter, alcohol (eg, behenyl alcohol), stearate (eg, glycerol monostearate), chelating agents (eg, EDTA), propylene glycol, SEPIGEL (Seppic, Inc., Fairfield, New York). Jersey, USA), silicone and silicone derivatives (eg dimethicone, cyclomethicone), vitamins (eg vitamin E), among others.

Buccal administration

The pharmaceutical composition according to the present invention may be prepared, packaged or sold in a composition suitable for buccal administration. Such compositions may, for example, take the form of tablets or lozenges prepared using conventional methods, and may, for example, contain from 0.1 to 20% (w/w) of the active ingredient, containing the remainder of the soluble or orally degradable composition, and optionally supplemented with one or more additional ingredients described herein. Alternatively, preparations suitable for buccal administration may contain a powder or aerosol or solution or suspension containing the active substance. Such powder, aerosol or aerosol compositions, when sprayed, preferably have an average particle size or droplet size in the range of from about 0.1 to about 200 nm, and may additionally contain one or more of the additional ingredients described herein.

Rectal administration

The pharmaceutical composition according to the present invention may be prepared, packaged or sold in a composition suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention microenema, and a colon hydrotherapy solution.

Suppositories can be prepared by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at normal room temperature (i.e., about 20°C) and which becomes liquid at the subject's rectal temperature (i.e., about 37°C in a healthy person). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycol, and various glycerides. Suppositories may additionally contain various additional ingredients, including, but not limited to, antioxidants and preservatives.

Micro enema retention compositions and colon hydrotherapy solutions can be prepared by combining the active substance with a pharmaceutically acceptable liquid carrier. As is well known in the art, drugs may be administered using an enema, and may be placed internally by a device adapted to the subject's rectal anatomy. The enema composition may further contain various additional ingredients including, but not limited to, antioxidants and preservatives.

Additional dosage forms

Additional dosage forms of the present invention include dosage forms as described in US Patent No. 6340475; 6488962; 6451808; 5972389; 5,582,837 and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in US Patent Applications Nos. forms of this invention also include dosage forms as described in PCT applications for No. 03/35041 WO, WO 03/35040, 03/35029 WO, WO 03/35177, 03/35039 WO, WO 02/96404, 02/32416 WO, WO 01/97783, 01/56544 WO, WO 01/32217 , 98/55107 WO, WO 98/11879, 97/47285 WO, WO 93/18755, and WO 90/11757.

Controlled Release Compositions and Drug Delivery System

Controlled or sustained release formulations of a pharmaceutical composition according to the present invention can be made using well known techniques such as the use of proteins with pH sensitive domains or protease cleavable moieties. In some cases, the dosage forms used may result in a slow or controlled release of one or more active ingredients in them using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes or microspheres or their combinations to provide the desired release profile in various proportions. Any controlled release formulations are known to those skilled in the art, including those described herein, and can be readily selected for use in a pharmaceutical composition in accordance with the present invention. Thus, single dose drug containers suitable for oral administration, such as tablets, capsules, gel capsules, and caplets that are adapted for controlled release, are within the scope of the present invention.

Most controlled release pharmaceutical compositions share the common goal of improving drug therapy compared to non-controlled release counterparts. Ideally, the use of optimally designed controlled-release formulations for drug therapy is characterized by a reduction in the drug used for treatment, or control of the condition in a minimum amount of time. The benefits of controlled release formulations include prolongation of drug action, reduced frequency of drug administration, and improved adherence to therapy. In addition, the controlled release compositions used affect the time of onset of action or other characteristics, such as blood levels of the drug, and thus the occurrence of side effects can be influenced.

Most controlled release formulations are designed to initially administer some amount of drug that promptly produces the desired therapeutic effect, and gradually and continuously administer other amounts of drug to maintain that level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

Controlled release of the active ingredient can be stimulated by various inducers, such as pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "controlled release of a component" in the context of the present invention is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes or microspheres, or a combination thereof, that facilitate the controlled release of the active ingredient.

In certain embodiments of the invention, compositions in accordance with the present invention may be, but not limited to, short-acting, accelerated release, as well as controlled, for example, sustained release, delayed release and pulsatile release.

The term "sustained release" is used in its conventional sense to refer to a dosage form that provides a gradual release of the drug over an extended period of time, and which may, although not necessarily, lead to a significant constant level of the drug in the blood over an extended period of time. The time period may be less than a month or longer and the release should be longer than with a bolus of the same amount of substance.

For sustained release, compositions can be formulated with a suitable polymer or hydrophobic agent to provide the composition with sustained release. As an example, the compositions used in the method of the present invention may be administered in the form of microparticles, for example by injection or as implantable plates or discs.

In a preferred embodiment, the compositions of the present invention are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

The term "sustained release" is used herein in its usual sense to refer to a dosage form for initially assimilating the drug some time after its administration and that this coating, although not necessarily, includes a delay of about 10 minutes to 12 hours.

The term pulsatile release is used herein in its usual sense to refer to a dosage form that releases a drug in such a way as to produce a pulsatile plasma profile of the drug after administration.

The term "immediate release" is used in its conventional sense to refer to a dosage form that releases the drug immediately after administration.

As used herein, short-term action refers to any period of time up to about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and with any integer or fractional step in between after drug administration.

As used herein, accelerated elimination is understood to mean any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and with any integer or fractional step in between after drug administration.

Accordingly, in one embodiment of the invention, the present invention relates to a chemical composition containing propanoic acid, isobutyric acid, at least one ester, and at least one carrier, and, the chemical composition has an antibacterial effect when applied to human or animal excrement. In another embodiment of the invention, at least one ester is isoamyl butyrate. In another embodiment of the invention, at least one carrier is a silica-based carrier. In another embodiment of the invention, at least one carrier is selected from the group consisting of bentonite, zeolite and perlite. In another embodiment, the ratio of propanoic acid: isobutyric acid: isoamyl butyrate is about 3.5:3.5:2 v/v/v. In another embodiment of the invention, the ratio of propanoic acid, isobutyric acid and isoamyl butyrate is about 7 parts of two acids and 2 parts of isoamyl butyrate. In another embodiment of the invention, the chemical composition comprises predominantly propanoic acid, isobutyric acid, isoamyl butyrate, and a carrier. In another embodiment of the invention, the carrier is selected from the group consisting of bentonite, zeolite and perlite. In another embodiment, the chemical composition has an antibacterial effect when applied to human and animal feces.

In another embodiment, the present invention relates to a chemical composition that contains propanoic acid, isobutyric acid, at least one ester, at least one carrier, and at least one fungus. In another embodiment of the invention, at least one ester is isoamyl butyrate. In another embodiment of the invention, at least one carrier is a silica-based carrier. In another embodiment of the invention, at least one carrier is selected from the group consisting of bentonite, zeolite and perlite. In another embodiment, the ratio of propanoic acid: isobutyric acid: isoamyl butyrate is about 3.5:3.5:2 v/v/v. isoamyl butyrate. In another embodiment of the invention, at least one fungus is an endophyte. In another embodiment, the endophyte belongs to the genus Fusarium . In another embodiment, the endophyte is F. subglutinans .

In another embodiment of the invention, the present invention relates to a chemical composition that contains propanoic acid and at least one ester containing an acidic residue with 6-12 carbon atoms, characterized in that the chemical composition has a propanoic acid: ester ratio of about 7: 2 volume/volume. In another embodiment of the invention, at least one ester is isoamyl hexanoate. In another embodiment of the invention, the composition further comprises at least one nutritional supplement and at least one salt. In another embodiment, the composition contains glucose, whey protein, potassium chloride, magnesium sulfate, and sodium chloride. In another embodiment, the composition contains glucose, glycine, potassium chloride, sodium chloride and magnesium acetate. In another embodiment, the composition contains glucose, glycine, potassium chloride, sodium chloride, magnesium acetate, and potassium monophosphate. In another embodiment of the invention, the composition contains at least one pharmaceutically acceptable carrier. In another embodiment of the invention, the carrier is a cremophor. In another embodiment, the composition contains predominantly propanoic acid and isoamyl hexanoate in a propanoic acid:isoamyl hexanoate ratio of about 7:2 v/v. In another embodiment of the invention, the present invention relates to a chemical composition that contains predominantly propanoic acid, isoamyl hexanoate and a carrier.

In another embodiment of the invention, the present invention relates to a chemical composition that contains propanoic acid and an ester containing an acidic residue with one carbon atom, characterized in that the chemical composition has a propanoic acid:ester ratio of about 7:2 v/v. In another embodiment of the invention, at least one ester is isoamyl formate. In another embodiment, the composition contains predominantly propanoic acid and isoamyl formate in a propanoic acid:isoamyl formate ratio of about 7:2 v/v. In another embodiment of the invention, the composition contains at least one carrier. In another embodiment of the invention, at least one carrier is a silica-based carrier. In another embodiment of the invention, at least one carrier is selected from the group consisting of bentonite, zeolite and perlite. In another embodiment of the invention, the chemical composition contains predominantly propanoic acid, isoamyl formate and a carrier.

In another embodiment of the invention, the present invention includes a chemical composition that contains propanoic acid, isoamyl formate, and at least one fungus. In another embodiment, the propanoic acid:isoamyl formate ratio is about 7:2 v/v. In another embodiment of the invention, at least one fungus is an endophyte. In another embodiment, the endophyte belongs to the genus Fusarium . In another embodiment, the endophyte is F. subglutinans .

In another embodiment of the invention, the present invention relates to a method for treating human and animal feces, comprising the interaction of human and animal feces with a composition containing propanoic acid, isobutyric acid and at least one ester, characterized in that the composition kills or inhibits the growth of bacteria in excrement of humans and animals. In another embodiment of the invention, the present invention relates to a method for treating human and animal feces, comprising the interaction of human and animal feces with a composition containing propanoic acid, isobutyric acid, at least one ester and at least one fungus, characterized in that propanoic acid , isobutyric acid and at least one ester, kill or reduce the growth of bacteria in human and animal excrement, and at least one fungus increases the rate of decomposition of human and animal excrement. In another embodiment, the present invention relates to a method of eliminating or reducing microbial growth in a treatment area in a treatment area, comprising contacting the treatment site with a composition that contains propanoic acid and at least one ester in a propanoic acid:ester ratio of approximately 7:2, wherein the ester is isoamyl formate or isoamyl hexanoate and the composition kills or reduces the growth of bacteria in human and animal feces. In another embodiment of the invention, the present invention relates to a method for treating human and animal feces, comprising the interaction of human and animal feces with a composition that contains propanoic acid, isoamyl formate in a ratio of propanoic acid: isoamyl formate of about 7:2, and at least one fungus , characterized in that the mixture of propanoic acid and isoamyl formate kills or reduces the growth of microbes in human and animal excrement, and at least one fungus increases the rate of decomposition of human and animal excrement. In another embodiment of the invention, the present invention relates to a method of treating an animal having a disease or disorder associated with a microbial infection, which includes administering to the animal an effective amount of a composition containing at least one organic acid. In another embodiment of the invention, the composition contains predominantly an organic acid. In another embodiment of the invention, the composition contains an organic acid. In another embodiment of the invention, at least one organic acid is propanoic acid. In another embodiment of the invention, at least one organic acid is isobutyric acid. In another embodiment of the invention, the animal is a human. In another embodiment, the disease or disorder is a gastrointestinal disease. In another embodiment of the invention, the animal is a domestic bull, pig or sheep. In another embodiment, the disease or disorder is selected from the group consisting of gastrointestinal diseases and intramammary infections. In another embodiment, the gastrointestinal disease is diarrhea. In another embodiment, the intramammary infection is subclinical or clinical mastitis. In another embodiment of the invention, the composition further comprises at least one ester. In another embodiment of the invention, at least one ester is isoamyl hexanoate. In another embodiment of the invention, the composition further comprises at least one nutritional supplement and at least one salt. In another embodiment, the composition contains glucose, whey protein, potassium chloride, magnesium sulfate, and sodium chloride. In another embodiment, the composition contains glucose, glycine, potassium chloride, sodium chloride and magnesium acetate. In another embodiment, the composition contains glucose, glycine, potassium chloride, sodium chloride, magnesium acetate, and potassium monophosphate. In another embodiment of the invention, the composition further comprises at least one pharmaceutically acceptable carrier. In another embodiment of the invention, the carrier is a cremophor. In another embodiment, the composition contains propanoic acid and isoamyl hexanoate in a propanoic acid:isoamyl hexanoate ratio of about 7:2 v/v. In another embodiment of the invention, at least one ester is isoamyl hexanoate. In another embodiment, the ratio of propanoic acid: isobutyric acid: isoamyl hexanoate is about 3.5:3.5:2 v/v/v. In another embodiment of the invention, the ratio of propanoic acid, isobutyric acid and isoamyl hexanoate is about 7 parts of two acids and 2 parts of isoamyl butyrate.

In another embodiment of the invention, the present invention relates to a chemical composition that contains predominantly propanoic acid, isobutyric acid, isoamyl hexanoate and a carrier. In another embodiment of the invention, the carrier is selected from the group consisting of bentonite, zeolite and perlite. In another embodiment, the chemical composition has an antibacterial effect when applied to human and animal excrement.

Those skilled in the art will recognize, or ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents have been contemplated within the scope of the present invention and are covered by the appended claims. For example, it should be understood that modifications to reaction conditions, including but not limited to reaction time, reaction size/volume, and experimental materials such as solvents, catalysts, pressure, conditions such as nitrogen atmosphere, and reducing/oxidizing agents, are recognized in the art by alternative techniques and using no more than routine experimentation are within the scope of this application.

It should be understood that wherever values and ranges are provided herein, all values and ranges do not cover those values and ranges that should be covered within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of the range of values, are also considered in this application.

The following examples further illustrate aspects of the present invention. However, they are in no way limited to the knowledge or descriptions of the present invention as set forth herein.

EXPERIMENTAL EXAMPLES

Hereinafter, the present invention will be described with reference to the following examples. These examples are provided for the purpose of illustration, and the present invention is not limited to these examples, but rather covers all variations that are apparent as a result of the information provided herein.

Example 1

The various experiments discussed below have led to the discovery of F. subglutinans and systems 1 and 2, which can be used in a variety of human and animal excreta handling devices, as well as animal bedding, stalls, etc.

Table 1. Shows the inhibitory activity of various esters (used in combination with a mixture of isobutyric and propanoic acids 1:1 v/v and in the same way a mixture of two acids is combined with an ester in a ratio of 7:2 v/v) in a series of tests on fungi and bacteria commonly used to determine antibiotic activity. Based on these data, systems 1 and 2 were selected for use in this invention (see highlighted areas in the table).

Table 2. For various newly isolated Fusarium spp growing on human feces in the presence of system 2 (with bentonite or zeolite as a carrier), molecular genetic data were obtained. It is therefore clear that either of these organisms is nearly equal to, or in most cases better than, Fusarium culmorum (P2-24), the subject of an earlier patent. Data array P2-24 is also included.

Table 3. Growth of various Fusariums on human feces results in a decrease in dry weight at week 7 of the experiment. The experimental setup contained 0.5 g bentonite with system 2 on a petri dish with medium containing about 100 mg wet weight human feces and a small agar block with test Fusarium . The incubation period was 7 weeks at 22°C. Residual human feces were physically removed and dried for 4 hours at 80°C and then weighed.

Fig. 1. Demonstrated what analyzes were done to obtain the data set in Table 1. For testing, various esters were combined with a mixture of isobutyric and propanoic acids 1:1 (v/v) and this mixture was added to the ester in a ratio of 7:2 v/v volume. Thereafter, 9 μl was placed in the central well with individual test organisms in agar blocks on the periphery, as shown in the figure.

Fig. 2. System 1 (above) has been shown to be effective in killing and inhibiting the growth of bacteria associated with human feces. Fresh human feces were collected and then about 5 mg was evenly distributed on the surface of the Potato Dextrose Agar Petri dish. Petri dishes were incubated for 2 days and then photographed. The petri dish on the right is the untreated control, the middle one contains 0.5 g of bentonite without antibiotic, and the left one contains 0.5 g of system 1.

Fig. 3. System 2 (above) has been shown to be effective in killing and inhibiting the growth of bacteria associated with human feces. Fresh human feces were collected and then about 5 mg was evenly distributed on the surface of the Potato Dextrose Agar Petri dish. Petri dishes were incubated for 2 days and then photographed. The petri dish on the right is the untreated control, the middle one contains 0.5 g of bentonite without antibiotic, and the left one contains 0.5 g of system 2.

Fig. 4. Illustrates two cat litter boxes with cat feces in each from 5 different cats about 140 gr. The container on the right was treated with System 1 on bentonite (0.5 ml/100 g bentonite). After 5 days ammonia measurements showed 14 ppm in the control container (left) and 0 ppm in the treated container. Overall, the odor was significantly reduced in the treated container.

Fig. 5. Demonstrated how the fungus can grow on fresh human feces and reduce odor levels. Treatment of about 140 g of human feces in the presence of urine with Fusarium subglutinans 06-1 and the presence of system 2 (1 ml per 10 g of zeolite). After 3 weeks there was a significant growth of F. subglutinans (white mycelium in the right container). The ammonia level was 71.4 in the control container on the left and 12.1 in the treated container on the right.

Fig. 6. Growth of various newly isolated Fusarium spp has been demonstrated compared to that of F. culmorum (p-2-24) on human feces. Progressive growth of Fusarium spp on small pieces of about 100 mg (wet weight) pieces of human feces over many days is shown. Growth comparison of recently isolated and characterized Fusarium spp compared to P2-24 is Fusarium culmorum , the subject of an earlier patent. New Fusarium spp. in particular, E06-1 and E06-5 do grow faster on human feces. Growth was measured by the radius of mycelium removal from an agar block placed on a piece of human excrement.

Fig. 7. Above, a six-day culture of the fungus Fusarium subglutinans E06-1, which is preferably used for the treatment of human and animal excrement in combination with system 2. Below, light microscopy of spores and F hyphae. subglutinans (bottom). The spores are slightly curved and have 9.8 -12×2.5 µm.

Fig. 8. Fusarium subglutinans (E06-8) grows vigorously on human feces (center) in the presence of System 2. Note the inhibition of bacterial growth on the right side of the culture dish, which is affected by System 2 fumes from bentonite particles on the left side of the dish. that promotes fungal growth. There, 0.5 g of treated bentonite was added, about 100 mg of human feces were added, and Petri dishes were incubated for 12 days. See fig. 6 with comparative height measurements.

Method 1 - method of experimental isolation of endophytes Fusarium spp

Isolates of Fusarium spp. can be obtained according to a standard method understandable to experts in this field of technology. Briefly, the shoot fragments were thoroughly wetted with 70% aqueous ethanol to disinfect the surface, and then the primary cortex/epidermis was removed with a sterile scalpel. Small fragments of aseptic secondary cortex were transferred to the surface of water agar (WA) and glycerol-arginine nutrient medium (GAS). After incubation for several days at 25°C, the ends of the hyphae developing fungi were aseptically removed and placed on potato dextrose agar (PDA). Pure fungal cultures were obtained in this manner. In particular, pink to red isolates with crescent-shaped spores are most likely endophytic Fusarium spp. Further characterization can be done using molecular techniques as will be appreciated by those skilled in the art. This procedure was used to search for each of the organisms used and described in this document.

Table 1 shows the inhibitory and destroying effect of propanoic acid and isobutyric acid together and separately and with various esters. The tests were carried out for 30 hours at 22 ^° C. The measurements were carried out with appropriate precautions and thus the percentage of growth inhibition for treated and control organisms (untreated) can be calculated. Bacteria and yeast-like organisms were evaluated based on relative growth rates after 30 h. These highlighted areas in the table show the compounds (esters) having the highest compatibility with the 1:1 (v/v) mixture of isobutyric and propanoic acids with the corresponding esters in a ratio of 7 to 2, systems 1 and 2 described above. It is through these tests that systems 1 and 2 were discovered. Acids are added at 7 µl separately and combined with esters to reach a level of 9 µl in the Petri dish test.

Table 2 provides a description of the molecular genetic data (below) obtained from new strains of Fusarium that have been tested for their ability to degrade human feces. Each of these isolates is designated as a heading. Detailed information on data acquisition is provided at the end of the table.

Table 2.

E 06-05 Fusarium subglutinans

Sequence (480 bases):

AACATACCAATTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGGGACGGCCCGCCAGAGGACCCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCC CGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCAGCTTGGTGTTGGACTCGCGAGTCAAATCGCGTTCCCCAAATTGATTGGCGGTCACGTCGAGCTTCCATAGCGTAGTAGTAAAACCCTCGTTACTGGTAATCGTCGCGGCACGCCGTTAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTA AGCATATCAATAA (SEQ ID NO: 1)

NCBI BLAST Compliance

Description Maximum value Total Leveling Weight Leveling coverage,% E-level Maximum match percentage Sequence identification code 18S rRNA gene of Fusarium verticillioides strain passport UOA/HCPF 14862 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% KC709665.1 Internal transcribed spacer 1 of strain 186_Jm Fungal sp. AM2013 (partial sequence); 5.8S rRNA gene and internal transcribed spacer 2, (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% KC506334.1 Fusarium subglutinans strain H1 18S rRNA gene 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% JX960431.1 Internal transcribed spacer 1 Fusarium sacchari (partial sequence); 5.8S rRNA gene and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% JN997445.1 The 18S rRNA gene of Fusarium sp. PRE4b (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|KC254039.1|), Gibberella intermedia culture collection 18S rRNA gene UOA/HCPF<GRC>: 12610, (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% JN254793.1 Gibberella moniliformis isolate FM2 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% JF499676.1 Genes 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA Gibberella moniliformis , strain MAFF 240085 (partial and complete sequence) 887 887 100% 0.0 100% AB587012.1 Genes 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA, Gibberella moniliformis strain CBS 576.78 (partial and complete sequence) 887 887 100% 0.0 100% AB587010.1 Genes 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA Fusarium subglutinans , strain ATCC 38016 (partial and complete sequence) 887 887 100% 0.0 100% AB587008.1 Gibberella moniliformis Gm3 strain 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% HQ718417.1

E 06-08 Fusarium subglutinans

Sequence (478 bases):

CATACCAATTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGGGACGGCCCGCCAGAGGACCCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCG CCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCAGCTTGGTGTTGGGACTCGCGAGTCAAATCGCGTTCCCCAAATTGATTGGCGGTCACGTCGAGCTTCCATAGCGTAGTAGTAAAACCCTCGTTACTGGTAATCGTCGCGGCACGCCGTTAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAG CATATCAATAA (SEQ ID NO: 2)

NCBI BLAST matches:

Description Maximum weight of the leveling section Total Leveling Weight Leveling coverage,% E-level Maximum match percentage Sequence identification code 18S rRNA gene of Fusarium verticillioides strain passport UOA/HCPF 14862 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 883 883 100% 0.0 100% KC709665.1 Internal transcribed spacer 1 of strain 186_Jm Fungal sp. AM2013 (partial sequence); 5.8S rRNA gene and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence 883 883 100% 0.0 100% KC506334.1 Internal transcribed spacer 1 of strain 165_Gbp Fungal sp. AM2013 (partial sequence); 5.8S rRNA gene and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 883 883 100% 0.0 100% KC506316.1 Fusarium subglutinans strain H1 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 883 883 100% 0.0 100% JX960431.1 Internal transcribed spacer 1 Fusarium sacchari (partial sequence); 5.8S rRNA gene and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 883 883 100% 0.0 100% JN997445.1 Internal transcribed spacer 1 of Gibberella moniliformis isolate FM11 (partial sequence); 5.8S rRNA gene and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 883 883 100% 0.0 100% HQ995666.1 The 18S rRNA gene of Fusarium sp. PRE4b, (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|KC254039.1|), 18S rRNA gene of Gibberella intermedia culture collection UOA/HCPF<GRC>:12610 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 883 883 100% 0.0 100% JN254793.1 Gibberella moniliformis isolate FM2 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 883 883 100% 0.0 100% JF499676.1 Genes 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA Gibberella moniliformis strain: MAFF 240085 (partial and complete sequence) 883 883 100% 0.0 100% AB587012.1 Genes 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA Gibberella moniliformis , strain: CBS 576.78 (partial and complete sequence) 883 883 100% 0.0 100% AB587010.1

E4-5 Fusarium sp.

Sequence (488 bases):

CTTAATTGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAACGGGACGGCCCGCCAGAGGACCCAAACTCTAATGTTTCTTATTGTAACTTCTGAGTAAAACAAACAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCT GGTATTCCGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCCCGGGTTTGGTGTTGGGGATCGGCCTCTGCCTTCTGGCGGTGCCGCCCCCGAAATACATTGGCGGTCTCGCTGCAGCCTCCATTGCGTAGTAGCTAACACCTCGCAACTGGAACGCGGCGCGGCCATGCCGTAAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATA CCCGCTGAACTTAAGCATATCAATAG (SEQ ID NO: 3)

NCBI BLAST matches:

Description Maximum weight of the leveling section Total Leveling Weight Leveling coverage,% E-level Maximum match percentage Sequence identification code 18S rRNA gene of non-cultivated clone R1_12 Fusarium (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 900 900 99% 0.0 100% KC753424.1 Genomic DNA containing the 18S rRNA gene, ITS1, the 5.8S rRNA gene, ITS2, and the 28S rRNA gene of an uncultivated Fusarium RRA10 clone, 900 900 99% 0.0 100% HE977525.1 Fusarium tricinctum isolate XSCZ07 internal transcribed spacer 1 (partial sequence); 5.8S rRNA gene and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 900 900 99% 0.0 100% JQ676180.1 Internal transcribed spacer 1 of a non-cultivated clone Hyp12 fungus (partial sequence); 5.8S rRNA gene and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 900 900 99% 0.0 100% JQ618507.1 18S gene, internal transcribed spacer 1, 5.8S rRNA gene, internal transcribed spacer 2, and region of the 28S rRNA rRNA gene of Fusarium tricinctum isolate UASWS0796 900 900 99% 0.0 100% JN662408.1 Internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and the 28S rRNA gene (partial sequence) of Fusarium sp. NRRL 52933 900 900 99% 0.0 100% JF740937.1 Internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence) Fusarium sp. NRRL 52714; and 28S rRNA gene (partial sequence) 900 900 99% 0.0 100% JF740911.1 Internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence) Fusarium sp. NRRL 25129; and 28S rRNA gene (partial sequence >gb|JF740916.1)|, internal transcribed spacer 1 of Fusarium sp. NRRL 52726, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|JF740917.1)|, internal transcribed spacer 1 of Fusarium sp. NRRL 52727, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and the 28S rRNA gene (partial sequence >gb|JF740918.1|) internal transcribed spacer 1 of Fusarium sp. NRRL 52730, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 900 900 99% 0.0 100% JF740895.1 Internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 of Fusarium sp. NRRL 25128 (full sequence); and 28S rRNA gene (partial sequence) 900 900 99% 0.0 100% JF740894.1 18S rRNA gene (partial sequence) of Gibberella avenacea isolate 3214; internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 900 900 99% 0.0 100% FJ224099.1

PC-2-24 (Control) Fusarium culmorum

Sequence (477 bases):

CATACCTTATGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAAAGGGACGGCCCGCCGCAGGAACCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTATAAAAAACAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCC CGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCAGCTTGGTGTTGGGAGCCTGCAGTCCTGCTGCACTCCCCAAATACATTGGCGGTCACGTCGAGCTTCCATAGCGTAGTAATTTACATATCGTTACTGGTAATCGTCGCGGCACGCCGTTAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCAT ATCAATAG (SEQ ID NO: 4)

NCBI BLAST matches:

Description Maximum weight of the leveling section Total Leveling Weight Leveling coverage,% E-level Maximum match percentage Sequence identification code Internal transcribed spacer 1 Fusarium sp. OTU930 (partial sequence); 5.8S rRNA gene and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 881 990 100% 0.0 100% GU934527.1 18S rRNA gene of Fusarium culmorum isolate 149 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 880 880 99% 0.0 100% KC989094.1 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA genes (partial and complete sequence) of Fusarium cerealis strain MAFF 101144 880 880 99% 0.0 100% AB820718.1 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA genes Fusarium culmorum strain MAFF 241212 >dbj|AB820717.1| (partial and complete sequence), genes containing 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA (partial and complete sequence) of Fusarium cerealis strain MAFF 241212 880 880 99% 0.0 100% AB586990.1 18S rRNA gene, internal transcribed spacer 1, 5.8S rRNA gene, internal transcribed spacer 2, and 28S rRNA gene region of Fusarium cerealis strain FC3 880 880 99% 0.0 100% JF303876.1 18S rRNA gene, internal transcribed spacer 1, 5.8S rRNA gene, internal transcribed spacer 2, and 28S rRNA gene region of Fusarium cerealis strain FC2 880 880 99% 0.0 100% JF303871.1 18S rRNA gene, internal transcribed spacer 1, 5.8S rRNA gene, internal transcribed spacer 2, and 28S rRNA gene region of Fusarium cerealis strain FC1 880 880 99% 0.0 100% JF303867.1 Fusarium culmorum strain G5 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 880 880 99% 0.0 100% GU566271.1 Internal transcribed spacer 1 of an uncultured clone B2_i_ITS1F Hypocreales (partial sequence); 5.8S rRNA gene (full sequence); and internal transcribed spacer 2 (partial sequence) 880 880 99% 0.0 100% EU754930.1 Internal transcribed spacer 1 of an uncultivated clone B3_1_c_ITS1F Hypocreales (partial sequence); 5.8S rRNA gene (full sequence); and internal transcribed spacer 2 (partial sequence) 880 880 99% 0.0 100% EU754928.1

E30-14 Fusarium avenaceum

Sequence (485 bases):

CAGAAGTTGGGGTTTTACGGCATGGCCGCGCCGCGTTCCAGTTGCGAGGTGTTAGCTACGCAATGGAGGCTGCAGCGAGACCGCCAATGTATTTCGGGGGCGGCACCGCCAGAAGGCAGAGCCGATCCCCAACACCAAACCCGGGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCGGAATACCAGCGGGCGCAATGTGCGTTCAA AGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTGTTTGTTTTACTCAGAAGTTACAATAAGAACATTAGAGTTTGGGTCCTCTGGCGGGCCGTCCCGTTTTACGGGGCGGGCTGATCCGCCGAGGCAACATTAAGGTATGTTCACAGGG GTTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGCA (SEQ ID NO: 5)

NCBI BLAST matches:

Description Maximum weight of the leveling section Total Leveling Weight Leveling coverage,% EE-level Maximum match percentage Identification code
sequences Identification code for sequences Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|JX402187.1|) 896 896 100% 0.0 100% JX402184.1 Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 896 896 100% 00.0 100% JX402183.1 Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 896 896 100% 0.0 100% JX402180.1 Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 896 896 100% 0.0 100% JX402179.1 Fusarium tricinctum wxm38 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 896 896 100% 0.0 100% HM037940.1 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA genes (partial and complete sequence) of Fusarium tricinctum isolate TS08-58-2 896 896 100% 0.0 100% AB470855.1 18S rRNA genes, ITS1, 5.8S rRNA, ITS2, 28S rRNA of Fusarium tricinctum isolate TS08-86-1 (partial and complete sequence >dbj|AB470818.1 |), 18S rRNA genes, ITS1, 5.8S rRNA, ITS2, 28S rRNA Fusarium tricinctum isolate TS08-70-1 (partial and complete sequence >gb|GU586834.1|), 18S rRNA gene of Fusarium tricinctum isolate Ppf30; internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 896 896 100% 0.0 100% AB470859.1 Gibberella avenacea isolate 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 896 896 100% 0.0 100% FJ602983.1 18S rRNA gene of Gibberella avenacea isolate FA18 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|FJ602975.1|), 18S rRNA gene of Gibberella avenacea isolate FA29 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|FJ603000.1|), 18S rRNA gene of Gibberella avenacea isolate FA54 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 896 896 100% 0.0 100% FJ602964.1 18S rRNA gene of Gibberella avenacea isolate FA17 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|FJ602968.1|), 18S rRNA gene Gibberella avenacea isolate FA22 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|FJ602973.1|), 18S rRNA gene of Gibberella avenacea isolate FA27 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|FJ602981.1|), 18S rRNA gene of Gibberella avenacea isolate FA35 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|FJ602999.1|), 18S rRNA gene of Gibberella avenacea isolate FA53 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 896 896 100% 0.0 100% FJ602963.1

E 06-7 Fusarium subglutinans

Sequence (469 bases):

CAGAAGTTGGGGTTTAACGGCGTGGCCGCGACGATTACCAGTAACGAGGGTTTTACTACGCTATGGAAGCTCGACGTGACCGCCAATCAATTTGGGGAACGCGATTTGACTCGCGAGTCCCAACACCAAGCTGGGCTTGAGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGA ATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTAGGGGTCCTCTGGCGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCAACAATTGGTATGTTCACAGGGGTTTGGGAGTTGTAA ACTCGGTAATGATTCCCTCCGC (SEQ ID NO: 6)

NCBI BLAST matches:

Description Maximum weight of the leveling section Total Leveling Weight Leveling coverage, % E-level Maximum match percentage Sequence identification code 18S rRNA gene of Fusarium verticillioides strain passport UOA/HCPF 14862 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 867 867 100% 0.0 100% KC709665.1 18S rRNA gene of Fusarium subglutinans strain AAFC-Fcir-012 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 867 867 100% 0.0 100% KC464632.1 Genomic DNA of Gibberella moniliformis strain DBT-112 containing the 18S rRNA gene, ITS1, the 5.8S rRNA gene, ITS2, and the 28S rRNA gene, 867 1017 100% 0.0 100% HF570008.1 Gibberella moniliformis isolate 18S rRNA gene SIDV20110221051 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 867 867 100% 0.0 100% KC143121.1 Internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 similar to the gene (partial sequence) Gibberella intermedia strain passport LFG4-3BBRS; mitochondrial 867 867 100% 0.0 100% JQ272470.1 Gibberella moniliformis strain CB1 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 867 867 100% 0.0 100% JX511973.1 The 18S rRNA gene of Fusarium sp. CHTAG40 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 867 867 100% 0.0 100% JF773630.1 The 18S rRNA gene of Fusarium sp. CHTAG38 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 867 867 100% 0.0 100% JF773629.1 The 18S rRNA gene of Fusarium sp. CHTAG34 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 867 867 100% 0.0 100% JF773628.1 The 18S rRNA gene of Fusarium sp. CHTAG32 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 867 867 100% 0.0 100% JF773627.1

E-30-7 Fusarium avenaceum

Sequence (480 bases):

GAAGTTGGGGGTTTTACGGCATGGCCGCGCCGCGTTCCAGTTGCGAGGTGTTAGCTACGCAATGGAGGCTGCAGCGAGACCGCCAATGTATTTCGGGGGCGGCACCGCCAGAAGGCAGAGCCGATCCCCAACCAAACCCGGGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCGGAATACCAGCGGGCGCAATGTGCGTTCAAAG ATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTGTTTGTTTTACTCAGAAGTTACAATAAGAAACATTAGAGTTTGGGTCCTCTGGCGGCCGTCCCGTTTTACGGGGCCGGGCTGATCCGCCGAGGCAACATTAAGGTATGTTCACAGGGG TTTGGGAGTTGTAAACTCGGTAATGATCCCTCC (SEQ ID NO: 7)

NCBI BLAST matches:

Description Maximum weight of the leveling section Total Leveling Weight Leveling coverage,% E-level Maximum match percentage Sequence identification code 18S rRNA gene of Fusarium avenaceum isolate 143 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% KC989099.1 18S rRNA gene of non-cultivated clone R1_12 Fusarium (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% KC753424.1 Fusarium avenaceum ribosome small subunit rRNA gene, Fk15 strain (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and the rRNA gene of the large subunit of the ribosome RNA (partial sequence) 887 887 100% 0.0 100% KC464345.1 Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|JX402187.1|), Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|JX402188.1|), Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|JX402189.1|), Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|JX402190.1)| Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% JX402184.1 Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% JX402183.1 Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% JX402180.1 Fusarium avenaceum 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% JX402179.1 Genomic DNA of an uncultivated Fusarium RRF01 clone, contains the 18S rRNA gene, ITS1, the 5.8S rRNA gene, ITS2, and the 28S rRNA gene 887 887 100% 0.0 100% HE977545.1 The 18S rRNA gene of Fusarium sp. CHTAM47 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% JF773662.1 The 18S rRNA gene of Fusarium sp. CHTAM2 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 887 887 100% 0.0 100% JF773634.1

E 06-01 Fusarium subglutinans

Sequence (468 bases):

GAAGTTGGGGTTTAACGGCGTGGCCGCGACGATTACCAGTAACGAGGGTTTTACTACGCTATGGAAGCTCGACGTGACCGCCAATCAATTTGGGGAACGCGATTTGACTCGCGAGTCCCAACACCAAGCTGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAAT TCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCAACAATTGGTATGTTCACAGGGGTTTGGGAGTTGTAAACT CGGTAATGATCCCTCCGCA (SEQ ID NO: 8)

NCBI BLAST matches:

Description Maximum weight of the leveling section Total Leveling Weight Leveling coverage, % E-level Maximum match percentage Sequence identification code 18S rRNA gene of Fusarium verticillioides strain passport UOA/HCPF 14862 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 865 865 100% 0.0 100% KC709665.1 The genomic DNA of Gibberella moniliformis strain DBT-112 contains the 18S rRNA gene, ITS1, the 5.8S rRNA gene, ITS2, and the 28S rRNA gene, 865 1016 100% 0.0 100% HF570008.1 Gibberella moniliformis isolate 18S rRNA gene SIDV20110221051 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 865 865 100% 0.0 100% KC143121.1 Gibberella moniliformis strain CB1 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 865 865 100% 0.0 100% JX511973.1 18S rRNA gene of Gibberella moniliformis isolate FM13 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 865 865 100% 0.0 100% HQ995667.1 Gibberella moniliformis isolate FM2 18S rRNA gene (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 865 865 100% 0.0 100% JF499676.1 Fusarium sp. Ljf001 18S rRNA gene, partial sequence; internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2, complete sequence; and 28S rRNA gene, partial sequence 865 865 100% 0.0 100% HQ025928.1 The 18S rRNA gene of the Gibberella sp. isolate FS-74(3) FLS-2010 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|HQ023214.1|), 18S rRNA gene of isolate FS-78 (3) Gibberella sp. FLS-2010 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence >gb|HQ023215.1|), 18S rRNA gene of isolate FS-55(3), Aspergillus sp. FLS-2010 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 865 865 100% 0.0 100% HQ023213.1 The 18S rRNA gene of the Gibberella sp. isolate FS-48.5(1) FLS-2010 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 865 865 100% 0.0 100% HQ023211.1 The 18S rRNA gene isolate FS-82(3) Gibberella sp. FLS-2010 (partial sequence); internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2 (full sequence); and 28S rRNA gene (partial sequence) 865 865 100% 0.0 100% HQ023203.1

Phylogenetic analysis based on ITS

Phylogenetic analysis of these organisms was carried out by studying the ITS sequences of the 5.8 S rRNA genes. Fungi were cultured on PDA for 7 days, and DNA templates were prepared using Prepman Ultra Sample Preparation Reagent (Applied Biosystems, USA) according to the manufacturer's recommendations. The fungal ITS regions were amplified with universal ITS primers ITS1 (5'TCCGTAGGTGAACCTGCGG 3'; SEQ ID no: 9) and ITS4 (5'TCCTCCGCTTATTGATATGC 3'; SEQ ID no: 10) using polymerase chain reaction (PCR). The PCR conditions were as follows: initial denaturation at 94°C for 3 min, then 30 cycles at 94°C for 15 sec, 50°C for 30 sec, 72°C for 45 sec, and final elongation at 72°C for 5 min. In 50 µl of the reaction mixture containing 1x PCR buffer, 200 µm of each dNTP, 1.5 mmol MgCl ₂ , 10 pmol of each primer, 1-5 ng of DNA and 2.5 units of Taq polymerase. The amplified product (5 μl) was visualized on a 1% (w/v) agarose gel to confirm the presence of one amplified band. The amplified products were purified on Amicon Ultra columns (Millipore, USA) and used at 20-40 ng per 10 µl sequencing reaction with the Big Dye Terminator sequencing kit (v. 3.1) with 2 pmol forward or reverse primer in the sequencing cycle reaction. Twenty cycles of 96°C for 10 sec, 50°C for 5°C and 60°C for 4 min were performed and the elongation products were purified by ethanol precipitation, dissolved in 10 µl of highly purified deionized formamide, incubated at 95°C for 4 min. for 1 min and loaded onto an I Prism 377 Genetic Analyzer (Perkin-Elmer, USA) for sequencing. All sequencing reagents were from Applied Biosystems, USA. The amplified products were sequenced and aligned with sequences in the GenBank database using the BLASTN program (Altschul et al., 1997). Sequencing was performed at the University of California, Berkeley.

As can be seen from Table 3, the growth of various fusariums on human feces leads to a decrease in dry weight at week 7 of the experiment. The experimental setup contained 0.5 g bentonite with system 2 on a Petri dish with medium containing about 100 mg wet weight human feces and a small agar block with test Fusarium . The incubation period was 7 weeks at 22°C. Residual human feces were physically removed and dried for 4 hours at 80°C and then weighed.

Table 3

Name of Fusarium isolates Dry weight of human faeces after 7 weeks, mg. Control is not Fusarium 43 EC-4-5 14 E06-7 23 E 06-7 25 P2-24 Fusarium culmorum control 24 E30-2 18 E30-7 27 E 06-1 16 E30-14 15 E06-8 14

Example 2

Development of a mixture of C-3 and C-4

To determine the biological activity of the various test mixtures against a panel of test microorganisms, tests were performed as described similarly for systems 1 and 2 above (FIG. 9). A small block with each microorganism was placed around the periphery of a Potato Dextrose Agar (PDA) Petri dish. The test solution was placed in the central well in a plastic cup holder. A control Petri dish (A) was also placed. After 30 hours of growth of test organisms compared to control, % inhibition was calculated. The Petri dish (B) contained the test mixture. Measurements were taken 30 hours after the Petri dish was placed.

It should be noted that the mixtures of systems 1 and 2 described herein contain about 3.5 parts of propanoic acid along with 3.5 parts of isobutyric acid and finally two parts of an ester of either isoamyl butyrate (system 1) or isoamyl isobutyrate (system 2 ). It is understood that while these mixtures are effective in a wide range of practical applications, there may be other mixtures that are even more effective in a large number of practical applications. To this end, a search was conducted using standard propanoic acid as a starting point while excluding isobutyric acid (due to its unpleasant odor), and now including high molecular weight esters as the ester component. Quite unexpectedly and unexpectedly, it was found that the use of seven parts of propanoic acid and two parts of isoamyl hexanoate formed a volatile mixture with biological activity exceeding B-23 (see below) and C-1, as shown in table 4. This new mixture is called as system 3, while the composition containing propanoic acid (7) and formate (2) is referred to as system 4. It should be noted that system 4 is less active against most of the tested microorganisms than system 3, but system 4 completely destroys E. coli, thereby allowing Fusarium to grow. For this purpose, system 4 is an effective mixture that can be used in the treatment of human excreta in conjunction with Fusarium spp.

Activity against yeast and bacteria

Yes = Height

Moderate = Some growth

None = No growth

* Testing C-3 was carried out for 30 hours at room temperature, and then measured and photographed. Measurements were taken from the edge of the inoculated block to the edge of the fungus colony. The two measurements taken were then averaged. The tests were carried out at room temperature. The results show that C-3 was the most biologically active mixture among the tested solutions. Also note C-3 inhibited Erwinia carotovora by 80-90% and inhibited Lactobacillus sp. on, about 50%.

** Test composition B-23 was: 1.39 parts of acetaldehyde; 2.83 parts of 2-butanone; 30.56 parts of propanoic acid, 2-methyl and methyl ester, 2.29 parts of acetic acid, 2-methylpropyl ester; 1.09 parts propanoic acid, 2-methyl-, 2-methylpropyl ester; 1.78 parts 1-propanol, 2-methyl-; 1.51 parts 2-butenal, 2-methyl-, (E)-; 4.79 parts 1-butanol, 3-methyl acetate; 4.78 parts propanoic acid 2-methyl-, 2-methylbutyl ester; 5.38 parts 1-butanol, 3-methyl-; 351.18 parts of propanoic acid, 2-methyl-; 1.31 parts of acetic acid, 2-phenylethyl ether.

It should be noted that the mixture of C-3 gave 100% inhibition of many tested microorganisms (table 4). The effect was higher than with isoamyl hexanoate or propanoic acid alone. Thus, in some cases, the mixture had a strong synergistic effect ie. 66% inhibition of Sclerotina sclerotiorum by hexanoate, 0% by propanoic acid and 100% by combining both components. Some other organisms react in the same way as Rhizoctonia solani . In addition, it turned out that C-3 was more active than C-1, as well as B-23 and, of course, isoamyl hexanoate or propanoic acid alone (table 4). Other combinations of propanoic acid with other esters or combinations of esters and terpenoids such as cineole or valencen were not as effective (Table 4). Composition C-4, although not as potent as C-3 and not as strong against Fusarium , does inhibit other microorganisms and thus may be better suited as an agent used in the treatment of human excreta in combination with Fusarium . In tests, C-3 kills both bacterial test organisms (Table 4). C-3 also acts Erwinia and Lactobacillus sp. (Table 4).

Determination of appropriate ratios of C-3 ingredients

Mixtures of propanoic acid in isoamyl hexanoate were varied and then tested according to the procedures outlined above. It turns out that in a mixture of two ingredients, the ratio of 7: 2 is most effective (Table 5). All other combinations showed lower rates of inhibition (table 5). The addition of terpenoids such as valensene did not increase biological activity. Thus, the ratio of the two components 7:2 volume/volume is the most preferred for practical use.

Yes = Height

Moderate = Some growth

None = No growth

C. Testing other esters with propanoic acid

As shown in Table 6 (below), alternative esters were tested mixed with propanoic acid in a ratio of 7:2 propanoic acid:ester. These compositions are in addition to compositions C-1, C-2, C-3 and C-4, and, accordingly, parts of the compositions in accordance with the present invention. It should also be understood that the present invention may include several esters or combinations of any of the esters described throughout this document in combination with propanoic acid, preferably in a ratio of 7:2 propanoic acid:ester.

Yes = Height

Moderate = Some growth

None = No growth

All tests were performed according to the methods in Table 4.

Example 3

Corn decontamination tests

Corn is fermented into ethanol. It was crushed, heated to a shapeless mass and treated with enzymes before adding yeast to the final preparation. One or more antibiotic preparations were also added, typically to suppress competing microorganisms otherwise disrupting the fermentation process. Since these antibiotics are being withdrawn from the market, other antimicrobials are needed. The following was done to determine if C-3 was effective against microorganism contamination in corn:

approximately 5 g of ground corn (crushed grain) was treated for 1 hour with 10 ml of 0% (control), 0.25%, 0.5% and 1% solutions of C-3, made in a ratio of 7: 2 v/v, plus 10 µl Triton-X-100 (in 10 ml). The treatment was carried out for 1 hour and the product was dried on a damp cloth to remove excess liquid. Approximately 2 g of material was placed directly in a PDA petri dish and incubated for 2 days before photographing. In another case, the crushed grains were dried under a hood and then sown on the PDA.

The results showed that treatment levels of 0.5% and 1.0% C-3 for 1 hour completely removed bacterial contamination of the crushed corn particles (FIG. 10). When the corn particles were dried and further dried and tested in the same way, the results were almost the same.

In general, the results obtained indicate that the C-3 solution can be used for disinfection in agriculture and food products and materials. This also applies to tools, equipment, equipment and foodstuffs processed for human consumption.

Example 4

The use of C-4 in the treatment of human feces with Fusarium subglutinans .

Compositions C-3 and C-4 were tested to determine their effectiveness in treating bacteria in human feces. Testing was carried out using 0.5 g of bentonite, in proportions of 1 ml of C-1, C-2, C-3 or C-4 or B-23 per 10 g of bentonite. The control did not contain mixtures of substances. Barley seeds infected with Fusarium subglutinans (the one used previously) were placed on PDA plates. In the middle of a Petri dish with bands of pure culture of E. coli obtained from human excrement. Covered and sealed with parafilm, then incubated for 3 days and photographed. C-3 and C-4 killed E. coli but were less effective than B-23. In the presence of C-3 , Fusarium grew better than with C-4. As shown in FIG. 11 E. coli grew in control and grew weakly in the presence of B-23. It should be noted that the C-4 in bentonite resulted in the complete death of E. coli , so the growth of Fusarium was observed. The same can be said for the treatment of C-1 and C-3, but the growth of Fusarium was inhibited to a greater extent. B-23 as a control was also effective in killing E. coli, but not to the same extent. The results show that C-4 is a good and reasonable preparation for treating human feces, killing intestinal bacteria with associated contaminants using Fusarium to decompose human feces.

Example 5

Treatment of C-3 animal feces

C-3 was applied at a rate of 3 ml per pound (453 g) of zeolite. The following experiments were carried out to test its effectiveness in treating animal excrement with control of bacterial growth. The treated zeolite (0.5 g) was placed in the central well of a PDA Petri dish (cut out). The Petri dish was completely covered with streaks and a suspension of bacterial cells obtained from chicken, cat, goat and horse excrement. Petri dishes were closed and sealed with parafilm, and then observed and photographed after 3-4 days of incubation. The results in all cases showed that C-3 is an effective antimicrobial mixture as it entails the formation of zones of inhibition on Petri dishes. This effect was also noted in the case of cat, goat and horse feces bacteria (Fig. 12). The results indicate that zeolite in combination with C-3 has potential for use in cat litter or chicken coop applications or as an animal bedding agent.

Example 6

Additional testing has been done on the effectiveness of C-3 in the treatment of cat litter and pet bedding.

Testing the Comparative Efficiency of Cat Litter Treatment

1 ft ^-2 plastic containers tightly closed on top were filled with bedding requiring treatment and untreated bentonite in the ratio indicated in the instructions on the package (figure 13). For these tests, CLOE (applied at 4 ml per pound (453 g)) and control untreated bentonite were tested. The test containers were maintained at a constant temperature throughout the experiment (eg, 70°F (21 ^° C)). Approximately 50 g of cat feces and 5 ml of urine were added to the treated containers. Ammonia levels were measured in each container at 24 hour intervals using a Z-800 gas analyzer. Measurements were made by placing the gas analyzer inside each box at appropriate 5-minute intervals, which minimized open container time. The ammonia analyzer reported the average ammonia level within five minutes, as well as the peak ammonia level reached within five minutes. After completion of measurements, one-day-old faeces were removed and about 50 g of fresh faeces and 5 ml of urine were added. The containers were closed. To determine the relative effectiveness of the funds, these actions were performed daily for 1 week. Once with an interval of one hour received data on the concentration of ammonia for each of the containers.

After 8 days, the average ammonia level read at 5-minute intervals every 24 hours showed that ammonia production in the CLOE-treated bedding was significantly lower than in the untreated control (see Figures 14A and 14B).

Example 7

Microbiological analysis

Microbiological studies have been carried out to demonstrate a direct relationship between odor generation processes and microbiological activity in waste and environmental waste. Suppression or control of microbial activity will have a beneficial effect on the reduction of unpleasant odors. CLOE has a direct effect on this microbial activity, instead of reducing the intensity of odors by absorbing them, as other treatments do. To demonstrate this quality, approximately 0.5 g of CLOE was placed in the center well of a Petri dish of potato dextrose agar (PDA) coated with a lawn of bacteria obtained from fresh hard cat feces. Petri dishes were incubated at 23°C for 1 week and photographed. The series of photographs shown in Figures 15A and 15B indicate that in the control bentonite experiment (15A) there were a huge number of bacterial colonies that grew on the Petri dish, including those areas close to the well containing bedding. In contrast, CLOE-treated (4 ml C-3 per pound (453 g) vehicle, 15B) Petri dishes were virtually free of bacterial colonies in all wells, but some growth of Penicillium sp. carrier and does not contribute to the formation of odor. Please pay attention to Penicillium sp. in the control well.

Clearly, the antimicrobial activity of CLOE is directly related to its effectiveness as a treatment for cat litter and its ability to reduce ammonia and other odors from animal (particularly cat) feces, as evidenced by many users. This also confirms the ability of the agent to inhibit and kill microorganisms associated with waste, such as E. coli . The CHLOE preparation is absorbed by the carrier but is also released slowly over time, and thus can be effective at some distance from a point source of the bentonite or zeolite carrier particle.

Example 8

Testing the Comparative Efficacy of Chicken Litter Improvers

1 ft ^-2 plastic containers tightly closed on top, filled with pine shavings and bedding recommended processed in the proportion indicated in the instructions on the package (Fig. 16). For these tests, Barnyard Bedding at 15 ml per pound (453 g) of zeolite and untreated zeolite (control) were tested, as these products represent a marketable commodity. The test containers were maintained at a constant temperature throughout the experiment (eg, 70°F (21 ^° C)). Approximately 50 g of chicken manure was added to the treated containers and sprinkled with approximately 2 ml of water. Ammonia levels were measured in each container at 24 hour intervals using a Z-800 gas analyzer. Measurements were made by placing the gas analyzer inside each box at appropriate 5-minute intervals, which minimized open container time. The ammonia analyzer reported the average ammonia level within five minutes, as well as the peak ammonia level reached within five minutes. After the measurements were completed, about 50 g of fresh manure and 2 ml of water were added every day and the containers were closed. To determine the relative effectiveness of the funds, these actions were performed daily for 3 weeks. Once with an interval of one hour received data on the concentration of ammonia for each of the containers.

After 8 days, average ammonia levels read at 5 minute intervals every 24 hours showed that ammonia production was highest in zeolite (control) and lowest in Barnyard Bedding (Figures 17A and 17B).

Example 9

Testing the Comparative Efficacy of Bedding Improvers in Cattle Stalls

1 ft ^-2 plastic containers tightly closed at the top, filled with pine shavings and bedding recommended processed in the proportion indicated in the instructions on the package (Fig. 18). For these tests, Barnyard Bedding and untreated zeolite (control) were tested. The test containers were maintained at a constant temperature throughout the experiment (eg, 70°F (21 ^° C)). Approximately 100 g of horse feces and 10 ml of urine were added to the treated containers. Ammonia levels were measured in each container at 24 hour intervals using a Z-800 gas analyzer. Measurements were made by placing the gas analyzer inside each box at appropriate 5-minute intervals, which minimized open container time. The ammonia analyzer reported the average ammonia level within five minutes, as well as the peak ammonia level reached within five minutes. After the measurements were completed, one-day-old faeces and urine-soaked pine chips were removed. Approximately 100 g of fresh manure and 10 ml of urine were added to the recommended proportion for the treatment of bedding with wet spots and each container was closed. To determine the relative effectiveness of the funds, these actions were performed daily for 1 week. Once with an interval of one hour received data on the concentration of ammonia for each of the containers.

After 8 days, an average ammonia level read at 5 minute intervals every 24 hours indicated that ammonia production was highest in the control litter and lowest in the Barnyard Bedding treated litter (Figures 19A and 19B).

Example 10

Treatment of diarrhea in a calf using composition C-3

Calf diarrhea is an acute intestinal infection in calves caused primarily by viral and bacterial infections of the calf. In some cases, diarrhea can occur in over 70% of the calves in a herd, and lead to the death of 50% of the infected calves. Although there is a viral etiology for such incidents, the most common cause is one of the more pathogenic strains of Escherichia coli bacteria, followed by strains of Cryposporidia and Salmonella .

Minimum inhibitory concentration (MIC) C-3 for E. coli, <0.00125%. See also Table 5 (above). Accordingly, a solution of approximately 1% C-3 in 50 ml may be effective in treating a calf suffering from diarrhea. Solution (C-X) was prepared for testing in calves diagnosed with classic symptoms of diarrhea.

Composition C-X contains the following ingredients:

For 100 ml:

1 g glucose

1 g whey protein

0.25 g KCl

0.25 g MgSO ₄

0.5 g NaCl in

1 ml C-3

Glucose and whey protein are added as a dietary supplement for treated animals, while other salts are added to improve the electrolyte balance of the animal. The added composition C-3 suppresses and kills pathogenic bacteria.

The first test was done on 5 newborn Holstein calves without diarrhoea, in order to find out if the C-X solution contains toxic or side-effect substances. Each healthy calf was injected with 50 ml of C-X solution and after 1 day and a few weeks later, no side effects were noted, and in particular there were no signs of chemical side effects or abnormal behavior.

Activities for testing the C-X solution on animals were carried out in vivo at the Bozeman Ranch in Montana. Diarrhea in animals (Aberdeen Angus) was first noted in cold wet weather a few weeks before the start of the experiments. Every animal with diarrhea had all the symptoms associated with the disease. Doses for each animal were set at 50 ml per animal per course of treatment.

Fifteen calves, which were authenticated as having diarrhea, were treated with 50 ml of the solution through the oral cavity using a tube. Thirteen calves treated with 1 dose recovered overnight. Two animals required a second dose of drug and recovered overnight after the second dose. In FIG. 20A shows one of two diarrheic calves (tag 166) that had to receive a second 50 ml of C-X treatment solution (images taken prior to administration of C-X solution). Note the large piles of excrement in the lower right corner and the drooping head and ears (Fig. 20A). One day after the second treatment with C-X solution, the calf was walking and free of diarrhea (FIG. 20B). The second day after the second treatment with C-X solution, the calf suckled its mother.

No deaths were recorded in the experiment. Ranchers have reported that the C-X solution is vastly superior to all other modern treatments they have used. Accordingly, C-X solution is a safe, rapid and effective treatment for diarrhea.

Example 11

Treatment of diarrhea in animal husbandry

A formulation for the treatment of diarrhea has been developed that contains compositions of C-3 with added sugar, amino acids, sodium and potassium chloride, and magnesium acetate. Many animals having infectious diarrhea (caused by a pathogen) have been cured. Infectious diarrhea usually involves yellow to brownish-greenish stools. This is evident if the parasite is the cause of the diarrhea, with blood in the feces. Non-infectious diarrhea (milky diarrhea) involves whitish feces. In this study, at least two animals had milky diarrhea and were not cured. In addition, it turned out that one animal had parasitic diarrhea and could not be cured either. In principle, all other animals (having a viral or bacterial cause of diarrhea) recovered after C-X treatment. In about 90% of cases, recovery occurred within 12-24 hours, with signs of recovery occurring within 3-4 hours. In some cases, the recovery took two days and required repeated administration of the drug. It's not like any other treatment. The material was delivered orally through a gastric tube or syringe. Other treatments with antibiotics and nutrient electrolyte solutions can help the animal, but recovery is not certain, as is the case with most C-3 treatments.

Exemplary compositions and treatment of diarrhea in calves

Based on 90 ml of water:

1 g glucose

1 g glycine

0.5 g NaCl

0.25 g KCl

0.25 g mg magnesium acetate

1 ml C-3 containing 0.7 ml propanoic acid and 0.2 ml isoamyl hexanoate.

Administer by syringe or gastric tube 50 ml per animal per course of treatment and if some animals do not recover within 24 hours, repeat treatment is necessary.

Exemplary compositions and treatment of diarrhea in piglets

Based on 90 ml of water:

1 g glucose

1 g glycine

0.5 g NaCl

0.25 g KCl

0.25 g magnesium acetate

0.1 g KN ₂ RO ₄

2 ml C-3 containing 0.7 ml propanoic acid and 0.2 ml isoamyl hexanoate.

Inject using a syringe 1-2 ml per piglet.

Field studies of the treatment of diarrhea with C-X solution

Case 1 - Ranch 1

May 16, 2014 - One calf developed diarrhea on May 6, treated with banomyn and two injections of LA-2009 (an anti-infective drug). 1 pint of electrolyte solution was also administered daily; however, the animal did not recover and suffered from 9 to 10 days of chronic diarrhea. On the morning of May 17, a calf with diarrhea was orally injected with 50 ml of C-X solution using a syringe. It was noted that the condition of the calf improved dramatically in the evening and on the morning of May 18, a complete recovery was noted. As of May 22, 2014, the calf had no signs of diarrhea. These results indicate that virtually all treatments for diarrhea failed and the animal did not enter a recovery phase until C-3 was administered.

Case 2 - Ranch 2

Calf diarrhea and deaths flooded Ranch 2 in the late winter of 2014. The C-X solution was provided to cattle farmers and they administered the solution to calves using gastric tubes to administer it. Three calves were treated in this way. The calves recovered from their diarrhea within 24 hours of treatment, and there were no other drugs known to be given concomitantly with C-X that resulted in recovery. Sick animals were sent to the barn and photographed in the process of their recovery. One such animal is shown in Fig. 21.

Case 3 - Ranch 3

Rancho 3 experienced a particularly cold winter during the calving season, including high winds and high amounts of snowfall. Under these conditions, diarrhea in calves is common.

Treatment of diarrhea in calves with C-X solution was carried out at the ranch from April 2 to April 24, 2014. Eleven animals were treated with 50 ml doses using gastric probing. The results were successful: one dose cured nine heads; while one animal underwent a second procedure and another animal required three separate procedures. In some cases, the use of C-X solution was not the only way of treatment. Some animals that also developed symptoms of pneumonia required Baytril, sulfa tablets, or Nuflor. All animals treated with C-X solution recovered, and the majority (9) recovered within 24 hours after the procedure.

Case 4 - Barn 1

May 15, 2014 - Barn1 is a production process for raising a Holstein dairy cow. This is a cowshed with 300 animals whose health and daily needs must be met. The barn demonstrated that the diarrhea was caused by rotavirus, which was pointed out by a veterinary center located next to their barn. 7 young animals with diarrhea were treated by prerol injection of C-X solution with a syringe. All animals treated with C-X solution recovered. All animals except one recovered within 24 hours, and one after repeated treatment with C-X. solution.

According to barn 1, in December 2013, 30 calves died of diarrhea, although these calves were treated with electrolytic solutions as well as antibiotics. Treatment of calves usually takes 5-7 days with repeated treatments, compared to 1 day and 1-2 doses of C-X treatment. In barn 1, oral administration of the drug was preferred and proved to be effective.

The operator of Cowshed 1 noted that "more is needed, as the substance really works." She was referring to the C-X solution and their success with this remedy. The barn will continue to treat diarrhea in calves with C-X technology, as well as provide information about animals that will subsequently be tested with the C-X drug.

Case 5 - Ranch 4

The C-X technology was also tested in a Hutterite pig production during the week after April 14, 2014, where hundreds of piglets were diagnosed with diarrhea. The presence of porcine epizootic diarrhea virus (EDV) in the pig population has been confirmed by the Newport Laboratory, Worthington, Minnesota. According to the manufacturer, more than 850 piglets have died in the previous three weeks, representing almost 100 percent mortality among infected piglets.

The manufacturer spoke about the results of his trials with the preparation for piglets using C-X technology in the treatment of diarrhea in pigs as of April 19, 2014. The manufacturer noted that around April 12, 2014, 8-day-old piglets with diarrhea were injected orally with a syringe of 6 ml of C-X. After 5 hours, the piglets improved and there were no signs of diarrhea the next day. Moreover, as of April 15, 10 piglets, all 14 days old, were suffering from diarrhea with the classic signs of dark yellow loose stools. These piglets were injected orally with a syringe with 4 ml of C-X solution and within 24 hours each animal became completely "dry". The manufacturer also indicated that, based on previous experience with the disease, he expected many deaths.

In addition, around April 19, ten 3-day-old piglets that showed signs of diarrhea were injected with a syringe of 2 ml of C-X solution. After a few weeks, all of these piglets were still alive. Thirty three-day-old piglets with signs of diarrhea were given another treatment. These 30 pigs were injected with 3 ml of C-X solution and all the pigs remained alive. Based on the manufacturer's previous experience, if left untreated, death is expected in almost all animals. The manufacturer also administered Thylane 40-50 to each study 3 day old pig that was treated between April 15th and 19th. Thylane is an antibiotic used to treat pneumonia. The manufacturer believed that Tilan-40 paired with C-X treatment is responsible for the survival of young animals; however, according to those skilled in the art, antibiotics are generally not effective against viral and bacterial intestinal infections. While not wishing to be bound by any particular theory, in this case Thylane-40 most likely did not have any effect on piglet survival.

Thanks to the C-X solution for the treatment of piglets, the producer did not lose animals from diarrhea. In addition, as of May 27, 2014, samples from piglets that were sent to the Norwalk laboratory after C-X treatment confirmed the absence of PED. This finding indicates that the C-X solution was successful in treating diarrhea in Harlovetown pigs. In addition, the disappearance of PEDV from the area may be related to C-X treatment, as all diarrheic piglets treated with this technology recovered.

Case 6 - Ranch 5

5 ranches near the Canadian border used C-X to treat diarrhea in their cattle ranch. Temperatures during the calving season were especially low during last winter with strong winds and snowfall. The C-X solution was provided at the beginning of April and April 6, 2014 was the first date of its application. Twenty-one animals were treated with C-X solution alone in the presence of diarrhea and the animals were treated with 50 ml of C-X solution via gastric tube. After 24 hours, 18 calves that were treated with C-X recovered; however, 3 animals required a second dose. These animals soon recovered after the course of treatment. Of the 21 treated animals, 8 had severe symptoms of pneumonia and were treated with Nuflor. The recovery of animals treated with C-X is not associated with the treatment of pneumonia. The last day of treatment was April 18, 2014. Overall, approximately 85 percent of animals treated with C-X technology recovered with a single dose of the solution. The results obtained show that C-X technology requires less time and is more effective than traditional forms of animal diarrhea treatment.

Case 7 - Ranch 6

Rancho 6 is a barn that used C-X technology to treat diarrhea in mid-April 2014. The farm has about 100 Holstein dairy cattle with various other animals, including beef cattle. The incidence of diarrhea on ranches is particularly high, and most animals develop diarrhea shortly after birth. The barn was provided with C-X and was administered to beef calves and Holstein calves at doses of 50 ml administered by syringe orally.

Initially, 8 calves were treated with C-X at doses of 50 ml via syringe, 7 of which presented with typical creamy yellow diarrhea and 1 calf (number 80) with white pasty diarrhea and after two days with watery diarrhea. This calf had characteristic milky diarrhea. This particular calf was also treated with C-X but then developed symptoms and died 4 days later. It was suspected, based on the characteristics of the diarrhea, that this particular calf's diarrhea was nutritionally induced, in which case C-X was not effective. Most of the animals that were treated with C-X solution recovered and made a full recovery within 24 hours; however, one Holstein calf took 48 hours to fully recover. In addition, one beef calf (number 34 as control) at the ranch, which showed signs of diarrhea and was not treated with C-X solution along with other animals, died within two weeks. Figure 23 is a series of images showing a calf treated with C-X before and after treatment.

Case 8 - Ranch 7

Rancher 7 was interested in using C-X technology in the early spring of 2014 to treat newborn calves with diarrhoea. The C-X solution was given to the owners and within a few weeks calves showing signs of diarrhea were immediately treated on the pasture with 50 ml of the C-X solution in two doses of 25 ml orally with a syringe. At least 15 calves were treated with C-X solution and each of them, except for one, recovered in 24 hours. One calf that did not recover within 24 hours had white stools that could be referred to as "milky diarrhoea". This calf also had pneumonia and was treated with high doses of Nuflor. Two additional treatments with C-X solution were also carried out. The calf recovered from the diarrhea and is now in good health. The owner noted that most often the calves recovered within 3-4 hours after the C-X solution injection procedure. The owner also noted that there was a cessation of "teeth grinding" in the calves, and a general improvement in the condition. The owner noted that the oral C-X syringe method is very easy to dose to calves weighing up to 100 pounds (45.35 kg). These results demonstrate that C-X administration can be performed using a gastric tube or syringe.

Case 9 - Ranch 8

Beef calves with diarrhea were tested at Ranch 8 at 4,459 feet from May 4, 2013 to May 21, 2014, predominantly in spring 2013 and winter and spring 2014. On this ranch, 300 calves are born in spring and autumn. One hundred and forty-two different calves were treated with CX solution or a combination of SX solution and other medications. The initial drug C-X was used until the end of January 2014, after which the improved drug C-X technology was used for the treatment of diarrhea.

Temperatures ranged from -20°F to 50°F (-6°C to 10 ^° C) during the months of February and March. Significant snowfalls and winds were observed during this period of time and cardinal temperature changes were recorded.

A large number of calves were observed clinical signs of diarrhea (protruding ears, sullen eyes, and profuse diarrhea). These calves were injected with 50 ml of C-X solution by gavage and checked about six hours later to determine if they had recovered or needed a second dose of the drug. Of the 243 treated calves, 36 were given either the solution alone, one or two doses, or a solution with a vitamin supplement added, while the rest were given standard antibiotics along with C-X treatment for diarrhea. Twelve calves received a second or third dose of C-X solution on the same day. In total, 243 treatments included a C-X solution, and 29 of which were administered either 2 or 3 times. The total number of calves treated with both drugs was 142. Drugs commonly used with C-X treatment included: Exid (treats respiratory infections), probiotics, Toxiban (absorbs toxins with charcoal), Oxytetracycline LA (antibiotic for conjunctivitis, foot rot and other infections), multivitamins, Inforce 3 (vaccine for the prevention of respiratory diseases), Draxxin (an antibiotic for the treatment of conjunctivitis, foot rot and other infections), and sulfa tablets (sulfonamides for antibacterial treatment).

Ranchers 8 were interviewed through March 3, 2014 about their experiences with C-X in treating diarrhea. Diarrhea was first reported in the calving season on February 27, 2014, with 40 doses of 50 ml each available. From February 27 to March 3, 15 calves with signs of diarrhea were treated with C-X technology. Of these 15 calves, 13 recovered overnight after a single dose, while 2 required a second dose and recovered overnight, with no deaths in any case. Half of the calves were treated in the pasture, only 6 were brought to the barn. Normal treatment for diarrhea includes electrolyte solutions, intravenous fluids, and some antibiotics such as tetracycline.

Producers noted that some calves were treated for pneumonia and given other medications; however, it has also been noted that C-X is far superior to other remedies and is credited with its use for a speedy and complete recovery. It has been estimated that without C-X, one third or more of the calves with diarrhea would have died. Additional observations of C-X treatment are that it is easy to administer and tolerated in the herd, that it is a quick treatment option, and does not require antibiotics if diarrhea is detected early enough. Figure 24 is a series of images showing a calf before and after C-X treatment.

Table 7 depicts the administration of C-X remedy to ranches 3, 5, and 8 without any additional preparations other than vitamin supplements.

Table 7: C-X Treatment without other drugs

Ranch C-X date Calf Treatment number 8 C-1 5/4/2013 Y122 1st 8 C-1 5/4/2013 Y306B 1st 8 C-1 5/6/2013 Y350a 1st 8 C-3 2/24/2014 Y3742 1st 8 C-3* 2/27/2014 Y26262 1st 8 C-3* 2/27/2014 Y321 1st 8 C-3* 2/27/2014 Y383 1st 8 C-3 3/2/2014 R18361 1st 8 C-3 3/2/2014 Y3730 1st 8 C-3 3/2/2014 Y387 1st 8 C-3 3/3/2014 G032 1st 8 C-3 3/3/2014 Y166461 1st 8 C-3 3/3/2014 Y402 1st 8 C-3 3/4/2014 G03 2nd 8 C-3 3/4/2014 G3731 1st 8 C-3 3/4/2014 R1396 1st 8 C-3 3/4/2014 Y29092 1st 8 C-3 3/5/2014 G309 1st 8 C-3 3/5/2014 Y29902 1st 8 C-3 3/5/2014 Y3311 1st 8 C-3 3/5/2014 Y8A20 1st 8 C-3 3/6/2014 R2451 2nd 8 C-3 3/6/2014 Y163391 1st 8 C-3 3/9/2014 Y2014 2nd 8 C-3 3/12/2014 R215 1st 8 C-3 3/15/2014 Y16629 3rd 8 C-3 3/15/2014 Y2014 2nd 8 C-3 3/16/2014 R26861 2nd 8 C-3 3/16/2014 Y2062 2nd 8 C-3 3/16/2014 Y3619 1st 8 C-3 3/17/2014 Y20060 2nd 8 C-3 3/18/2014 Y3630 2nd 8 C-3 3/18/2014 Y80842 2nd 8 C-3 3/27/2014 R616 2nd 8 C-3 3/27/2014 Y411 2nd 8 C-3 3/30/2014 R616 1st 5 C3 4/6/2014 516 1st 5 C3 4/6/2014 1122 1st 5 C3 4/6/2014 1255 1st 5 C3 4/6/2014 8104 1st 5 C3 4/6/2014 9203 1st 5 C3 4/7/2014 7490 1st 5 C3 4/8/2014 173 1st 5 C3 4/8/2014 1241 1st 5 C3 4/8/2014 5416 1st 5 C3 4/12/2014 1203 2nd 5 C3 4/12/2014 2487 1st 5 C3 4/12/2014 2637 1st 5 C3 4/16/2014 1219 2nd 5 C3 4/18/2014 711 1st 5 C3 4/18/2014 1000 1st 5 C3 4/18/2014 1636 1st 5 C3 4/18/2014 k23 1st 3 C-X 4/18/2014 2431 1st * Received a multivitamin preparation

Table 8 shows data for all C-X treatments regardless of whether any additional medications were administered in ranches 3, 5, and 8.

Table 8: All treatments C-X

Ranch C-X date Calf Treatment number Additional
medicinal
facilities 8 C-1 5/4/2013 Y122 1st 8 C-1 5/4/2013 Y306B 1st 8 C-1 5/6/2013 Y350a 1st 8 C-1 5/15/2013 Y163 1st Oxytetracycline LA, Toxiban, Nas 8 C-1 5/15/2013 Y169 1st Oxytetracycline LA 8 C-1 5/15/2013 Y3351 1st Oxytetracycline LA 8 C-1 5/18/2013 R22326 1st Oxytetracycline LA 8 C-1 5/18/2013 Y169 1st Oxytetracycline LA 8 C-1 5/18/2013 Y3351 1st Oxytetracycline LA 8 C-1 5/29/2013 Y2886 1st Oxytetracycline LA, Toxiban 8 C-1 10/21/2013 Y311 1st Exid 5 cu. cm 8 C-1 10/23/2013 R287 1st Oxytetracycline LA 5cc cm 8 C-1 10/25/2013 R325 1st Oxytetracycline LA 5cc cm 8 C-1 10/25/2013 Y238 1st Oxytetracycline LA 5cc cm 8 C-3 2/23/2014 G309 1st Oxytetracycline LA 5cc see, Toxiban, sulfamides 8 C-3 2/23/2014 Y124981 1st Oxytetracycline LA 5cc see, Toxiban, sulfamides 8 C-3 2/23/2014 Y14146 1st Oxytetracycline LA 5cc see, Toxiban, sulfamides 8 C-3 2/23/2014 Y17739 1st Oxytetracycline LA 5cc see, Toxiban, sulfamides 8 C-3 2/23/2014 Y36192 1st Oxytetracycline LA 5cc see, Toxiban, sulfamides 8 C-3 2/23/2014 Y3742 1st Oxytetracycline LA 5cc see, Toxiban, sulfamides 8 C-3 2/23/2014 Y405 1st Oxytetracycline LA 5cc see, Toxiban, sulfamides 8 C-3 2/23/2014 Y420 1st Oxytetracycline LA 5cc see, Toxiban, sulfamides 8 C-3 2/24/2014 Y3742 1st 8 C-3 2/27/2014 Y26262 1st Multivitamins 8 C-3 2/27/2014 Y321 1st Multivitamins 8 C-3 2/27/2014 Y336 1st Oxytetracycline LA, multivitamins 8 C-3 2/27/2014 Y383 1st multivitamins 8 C-3 2/27/2014 Y422 1st 8 C-3 3/2/2014 R18361 1st 8 C-3 3/2/2014 Y3730 1st 8 C-3 3/2/2014 Y387 1st 8 C-3 3/3/2014 G032 1st 8 C-3 3/3/2014 Y166461 2nd 8 C-3 3/3/2014 Y188352 1st Oxytetracycline LA, Toxiban, multivitamins 8 C-3 3/3/2014 Y402 1st 8 C-3 3/4/2014 G03 1st Oxytetracycline LA, Toxiban, multivitamins 8 C-3 3/4/2014 G03 2nd 8 C-3 3/4/2014 G3731 1st 8 C-3 3/4/2014 R1396 1st 8 C-3 3/4/2014 Y29092 1st 8 C-3 3/4/2014 Y389 1st Oxytetracycline LA, Toxiban 8 C-3 3/5/2014 G309 1st 8 C-3 3/5/2014 Y29902 1st 8 C-3 3/5/2014 Y3311 1st 8 C-3 3/5/2014 Y3779 1st Toxiban 8 C-3 3/5/2014 Y8A20 1st 8 C-3 3/6/2014 G0561 1st Toxiban 8 C-3 3/6/2014 G3731 1st Oxytetracycline LA, Toxiban 8 C-3 3/6/2014 R1396 1st Oxytetracycline LA, Toxiban 8 C-3 3/6/2014 R2451 1st Oxytetracycline LA, Toxiban 8 C-3 3/6/2014 R2451 2nd 8 C-3 3/6/2014 R2456 1st Toxiban 8 C-3 3/6/2014 Y163391 1st 8 C-3 3/6/2014 Y20251 1st Toxiban 8 C-3 3/6/2014 Y3859 1st Oxytetracycline LA, Toxiban 8 C-3 3/6/2014 Y408 1st Toxiban 8 C-3 3/7/2014 G03 1st Oxytetracycline LA 5cc cm 8 C-3 3/7/2014 G522 1st Oxytetracycline LA 5cc cm 8 C-3 3/7/2014 R22519 1st Oxytetracycline LA 5cc cm 8 C-3 3/7/2014 Y16131 1st Oxytetracycline LA 5cc cm 8 C-3 3/7/2014 Y1951 1st Oxytetracycline LA 5cc cm 8 C-3 3/7/2014 Y29961 1st Oxytetracycline LA 5cc cm 8 C-3 3/7/2014 Y29962 1st Oxytetracycline LA 5cc cm 8 C-3 3/7/2014 Y3779 1st Oxytetracycline LA 5cc cm 8 C-3 3/8/2014 R2451 1st Oxytetracycline LA 5cc cm 8 C-3 3/8/2014 Y28869 1st Oxytetracycline LA 5cc cm 8 C-3 3/8/2014 Y29962 1st Exid 3 cu. cm 8 C-3 3/9/2014 G235 1st Oxytetracycline LA 5cc cm 8 C-3 3/9/2014 R13951 1st Oxytetracycline LA 5cc cm 8 C-3 3/9/2014 Y163391 1st Oxytetracycline LA 5cc cm 8 C-3 3/9/2014 Y1954 1st Exid 3 cu. cm 8 C-3 3/9/2014 Y2014 2nd 8 C-3 3/9/2014 Y2014 1st Oxytetracycline LA 5cc cm 8 C-3 3/9/2014 Y2220 1st Oxytetracycline LA 5cc cm 8 C-3 3/9/2014 Y3016 1st Oxytetracycline LA 5cc cm 8 C-3 3/9/2014 Y3042 1st Oxytetracycline LA 5cc cm 8 C-3 3/9/2014 Y3562 1st Oxytetracycline LA 5cc cm 8 C-3 3/10/2014 Y166461 1st Toxiban 8 C-3 3/10/2014 Y19541 1st Oxytetracycline LA 5cc cm 8 C-3 3/10/2014 Y2366 1st Oxytetracycline LA 5cc cm 8 C-3 3/10/2014 Y28392 1st Toxiban 8 C-3 3/10/2014 Y2985 1st Oxytetracycline LA 8 C-3 3/10/2014 Y3630 1st Oxytetracycline LA 8 C-3 3/10/2014 Y365 1st Oxytetracycline LA 8 C-3 3/10/2014 Y401 1st Oxytetracycline LA 5cc cm 8 C-3 3/11/2014 R2391 1st Oxytetracycline LA 8 C-3 3/11/2014 Y2014 1st Oxytetracycline LA 5cc cm 8 C-3 3/11/2014 Y2162 1st Exid 3 cu. cm 8 C-3 3/11/2014 Y2985 1st Oxytetracycline LA 5cc cm 8 C-3 3/11/2014 Y2985 1st Oxytetracycline LA 5cc cm 8 C-3 3/11/2014 Y3042 1st Exid 3 cu. cm 8 C-3 3/12/2014 R215 1st 8 C-3 3/12/2014 Y19349 1st Oxytetracycline LA 5cc cm 8 C-3 3/12/2014 Y2116 1st Exid 8 C-3 3/12/2014 Y2366 1st Exid 5 cu. cm 8 C-3 3/12/2014 Y2985 1st Exid 8 C-3 3/12/2014 Y29962 1st Oxytetracycline LA 5cc cm 8 C-3 3/12/2014 Y3630 1st Exid 5 cu. cm 8 C-3 3/12/2014 Y64659 1st Oxytetracycline LA 5cc cm 8 C-3 3/13/2014 G3731 1st Oxytetracycline LA 5cc cm 8 C-3 3/13/2014 R215 1st Exid 5 cu. see, Toxiban 8 C-3 3/13/2014 Y16629 1st Oxytetracycline LA 5cc cm 8 C-3 3/13/2014 Y19349 1st Oxytetracycline LA 5cc cm 8 C-3 3/13/2014 Y28392 1st Exid 8 C-3 3/14/2014 Y1106 1st Oxytetracycline LA 5cc cm 8 C-3 3/14/2014 Y16629 1st Exid 3 cu. cm 8 C-3 3/14/2014 Y2951 1st Oxytetracycline LA 5cc cm 8 C-3 3/15/2014 R26861 1st Oxytetracycline LA 8 C-3 3/15/2014 R2902 1st Oxytetracycline LA 5cc cm 8 C-3 3/15/2014 Y16629 3rd 8 C-3 3/15/2014 Y2014 1st Oxytetracycline LA 8 C-3 3/15/2014 Y2014 2nd 8 C-3 3/15/2014 Y2366 1st The ex 8 C-3 3/15/2014 Y28392 1st Oxytetracycline LA 5cc cm 8 C-3 3/15/2014 Y2985 1st Draxin 8 C-3 3/15/2014 Y356 1st Oxytetracycline LA 8 C-3 3/15/2014 Y3619 1st Oxytetracycline LA 8 C-3 3/16/2014 R21441 1st Oxytetracycline LA 5cc see, Toxiban 8 C-3 3/16/2014 R26861 2nd 8 C-3 3/16/2014 Y2062 2nd 8 C-3 3/16/2014 Y2062 1st Oxytetracycline LA 5cc cm 8 C-3 3/16/2014 Y2382 1st Oxytetracycline LA 5cc cm 8 C-3 3/16/2014 Y264 1st Oxytetracycline LA 5cc cm 8 C-3 3/16/2014 Y3619 1st 8 C-3 3/17/2014 G235 1st Oxytetracycline LA 5cc cm 8 C-3 3/17/2014 G3731 1st Oxytetracycline LA 5cc cm 8 C-3 3/17/2014 R13951 1st Oxytetracycline LA 5cc cm 8 C-3 3/17/2014 R317 1st Oxytetracycline LA 5cc cm 8 C-3 3/17/2014 Y20060 2nd 8 C-3 3/17/2014 Y20060 1st Oxytetracycline LA 5cc cm 8 C-3 3/17/2014 Y2116 1st Oxytetracycline LA 5cc cm 8 C-3 3/17/2014 Y2382 2nd Oxytetracycline LA 5cc cm 8 C-3 3/17/2014 Y264 1st Oxytetracycline LA 5cc cm 8 C-3 3/17/2014 Y2951 1st Oxytetracycline LA 5cc cm 8 C-3 3/17/2014 Y3630 1st Oxytetracycline LA 5cc cm 8 C-3 3/17/2014 Y80842 1st Oxytetracycline LA 5cc cm 8 C-3 3/18/2014 G34491 1st Oxytetracycline LA 5cc cm 8 C-3 3/18/2014 G4335 1st Oxytetracycline LA 5cc cm 8 C-3 3/18/2014 Y3630 2nd 8 C-3 3/18/2014 Y64659 1st Oxytetracycline LA 5cc cm 8 C-3 3/18/2014 Y80842 2nd 8 C-3 3/19/2014 R215 1st Oxytetracycline LA 5cc cm 8 C-3 3/19/2014 R29092 1st Oxytetracycline LA 5cc cm 8 C-3 3/19/2014 Y2382 3rd Oxytetracycline LA 5cc cm 8 C-3 3/19/2014 Y2951 1st Oxytetracycline LA 5cc cm 8 C-3 3/19/2014 Y2985 1st Oxytetracycline LA 5cc cm 8 C-3 3/19/2014 Y3619 1st Oxytetracycline LA 5cc cm 8 C-3 3/19/2014 Y64659 2nd Oxytetracycline LA 5cc cm 8 C-3 3/19/2014 Y80842 3rd Exid 3 cu. cm 8 C-3 3/20/2014 Y2951 1st Oxytetracycline LA 5cc cm 8 C-3 3/20/2014 Y411 1st Oxytetracycline LA 5cc cm 8 C-3 3/20/2014 Y80B42 1st Exid 3 cu. cm 8 C-3 3/22/2014 R616 1st Oxytetracycline LA 5cc cm 8 C-3 3/22/2014 Y411 3rd Exid 3 cu. cm 8 C-3 3/23/2014 R20016 1st Oxytetracycline LA 5cc cm 8 C-3 3/23/2014 R616 2nd Oxytetracycline LA 5cc cm 8 C-3 3/23/2014 Y2382 1st Oxytetracycline LA 5cc cm 8 C-3 3/23/2014 Y2951 1st Oxytetracycline LA 5cc cm 8 C-3 3/24/2014 R1836 1st Oxytetracycline LA 5cc cm 8 C-3 3/24/2014 R29092 1st Oxytetracycline LA 5cc cm 8 C-3 3/24/2014 R616 3rd Oxytetracycline LA 5cc cm 8 C-3 3/24/2014 Y10651 1st Draksin, Enforce 3 8 C-3 3/24/2014 Y321 1st Draksin, Enforce 3 8 C-3 3/24/2014 Y3619 1st Oxytetracycline LA 5cc cm 8 C-3 3/24/2014 Y411 1st Oxytetracycline LA 5cc cm 8 C-3 3/25/2014 R1836 2nd Oxytetracycline LA 5cc cm 8 С-3* 3/25/2014 R290 1st Draksin, sulfamides 8 C-3 3/25/2014 R29092 1st Oxytetracycline LA 5cc cm 8 C-3 3/25/2014 Y29852 1st Oxytetracycline LA 5cc cm 8 C-3 3/25/2014 Y3619 2nd Oxytetracycline LA 5cc cm 8 С-3* 3/25/2014 Y368 1st Draksin, sulfamides 8 С-3* 3/25/2014 Y3742 1st Draksin, sulfamides 8 С-3* 3/25/2014 Y3862 1st Draksin, sulfamides 8 С-3* 3/26/2014 G1310 1st Draksin, sulfamides 8 С-3* 3/26/2014 R239 1st Draksin, sulfamides 8 С-3* 3/26/2014 R239 2nd Draksin, sulfamides 8 С-3* 3/26/2014 R3000 1st Draksin, sulfamides 8 С-3* 3/26/2014 R3041 1st Draksin, sulfamides 8 С-3* 3/26/2014 Y1606 1st Draksin, sulfamides 8 С-3* 3/26/2014 Y20142 1st Draksin, sulfamides 8 C-3 3/26/2014 Y259 1st Oxytetracycline LA 5cc cm 8 С-3* 3/26/2014 Y28869 1st Draksin, sulfamides 8 С-3* 3/26/2014 Y29062 1st Draksin, sulfamides 8 С-3* 3/26/2014 Y30162 2nd Draksin, sulfamides 8 С-3* 3/26/2014 Y30162 1st Draksin, sulfamides 8 С-3* 3/26/2014 Y331 1st Draksin, sulfamides 8 C-3 3/26/2014 Y3619 3rd Oxytetracycline LA 5cc cm 8 C-3 3/27/2014 R1836 1st Oxytetracycline LA 5cc cm 8 C-3 3/27/2014 R616 1st Oxytetracycline LA 5cc cm 8 C-3 3/27/2014 R616 2nd 8 C-3 3/27/2014 R8461 1st Draksin, sulfamides 8 C-3 3/27/2014 Y411 1st Oxytetracycline LA 5cc cm 8 C-3 3/27/2014 Y411 1st 8 С-3* 3/28/2014 Y1361 1st Draksin, sulfamides 8 С-3* 3/28/2014 Y1606 1st Draksin, sulfamides 8 C-3 3/28/2014 Y266962 1st Oxytetracycline LA 5cc cm 8 С-3* 3/29/2014 G1310 1st Draksin, sulfamides 8 С-3* 3/29/2014 R2028 1st Draksin, sulfamides 8 С-3* 3/29/2014 Y16932 1st Draksin, sulfamides 8 С-3* 3/29/2014 Y18835 1st Draksin, sulfamides 8 С-3* 3/29/2014 Y3590 1st Draksin, sulfamides 8 С-3* 3/30/2014 G3060 1st Draksin, sulfamides 8 C-3 3/30/2014 R616 1st 8 С-3* 3/30/2014 Y166461 1st Draksin, sulfamides 8 С-3* 3/30/2014 Y2116 1st Draksin, sulfamides 8 C-3 3/30/2014 Y259 1st Oxytetracycline LA 5cc cm 8 С-3* 3/31/2014 G131 1st Draksin, sulfamides 8 С-3* 3/31/2014 G5052 1st Draksin, sulfamides 8 C-3 3/31/2014 G57 1st Oxytetracycline LA 5cc cm 8 C-3 3/31/2014 R3691 1st Oxytetracycline LA 5cc cm 8 C-3 3/31/2014 Y1124 1st Oxytetracycline LA 5cc cm 8 С-3* 3/31/2014 Y119 1st Draksin, sulfamides 8 C-3 3/31/2014 Y26682 1st Oxytetracycline LA 5cc cm 8 С-3* 3/31/2014 Y2839 1st Draksin, sulfamides 8 С-3* 3/31/2014 Y36192 1st Draksin, sulfamides 8 С-3* 3/31/2014 Y365 1st Draksin, sulfamides 8 С-3* 3/31/2014 Y384 1st Draksin, sulfamides 8 C-3 3/31/2014 Y386 1st Oxytetracycline LA 5cc cm 8 C-3 3/31/2014 Y424 1st Oxytetracycline LA 5cc cm 8 С-3* 4/1/2014 B1395 1st Draksin, sulfamides 8 С-3* 4/1/2014 G5052 2nd Draksin, sulfamides 8 C-3 4/1/2014 Y1126 2nd Oxytetracycline LA 5cc cm 8 C-3 4/1/2014 Y2446 1st Oxytetracycline LA 5cc cm 8 C-3 4/1/2014 Y266962 2nd Oxytetracycline LA 5cc cm 8 C-3 4/1/2014 Y29852 1st Oxytetracycline LA 5cc cm 8 С-3* 4/1/2014 Y3279 1st Draksin, sulfamides 8 С-3* 4/1/2014 Y3412 1st Draksin, sulfamides 8 С-3* 4/1/2014 Y43 1st Draksin, sulfamides 8 С-3* 4/1/2014 Y592 1st Draksin, sulfamides 8 С-3* 4/2/2014 Y17739 1st Draxin 8 С-3* 4/2/2014 Y26262 1st Draksin, sulfamides 8 C-3 4/2/2014 Y29852 2nd Oxytetracycline LA 5cc cm 8 С-3* 4/2/2014 Y399 1st Draksin, sulfamides 8 С-3* 4/3/2014 Y121242 1st Draxin 8 С-3* 4/3/2014 Y16936 1st Draxin 8 С-3* 4/3/2014 Y411 1st Draxin 8 C-3 5/21/2014 G34491 1st Oxytetracycline LA 5cc cm 8 C-3 5/21/2014 R1836 1st Oxytetracycline LA 5cc cm 8 C-3 5/21/2014 R20016 1st Oxytetracycline LA 5cc cm 8 C-3 5/21/2014 R2616 1st Oxytetracycline LA 5cc cm 8 C-3 5/21/2014 R29092 1st Exid 3 cu. cm 8 C-3 5/21/2014 Y411 2nd Oxytetracycline LA 5cc cm 8 C-3 5/21/2014 Y5861 1st Oxytetracycline LA 5cc cm 5 C3 4/6/2014 8104 1st 5 C3 4/6/2014 9203 1st 5 C3 4/6/2014 1122 1st 5 C3 4/6/2014 516 1st 5 C3 4/6/2014 1255 1st 5 C3 4/7/2014 1255 2nd Nuflor 5 C3 4/7/2014 7490 1st 5 C3 4/8/2014 1203 1st Nuflor 5 C3 4/8/2014 1241 1st 5 C3 4/8/2014 5416 1st 5 C3 4/8/2014 173 1st 5 C3 4/12/2014 2363 1st Nuflor 5 C3 4/12/2014 9203 1st Nuflor 5 C3 4/12/2014 1203 2nd 5 C3 4/12/2014 2637 1st 5 C3 4/12/2014 2487 1st 5 C3 4/12/2014 2763 1st Nuflor 5 C3 4/14/2014 1219 1st Nuflor 5 C3 4/16/2014 1219 2nd 5 C3 4/18/2014 1000 1st 5 C3 4/18/2014 1636 1st 5 C3 4/18/2014 k23 1st 5 C3 4/18/2014 711 1st 3 C-X 4/8/2014 2012 1st Laxative 3 C-X 4/18/2014 Y273 1st Laxative, Baytril 3 C-X 4/18/2014 2431 1st 3 C-X 4/18/2014 Y177 1st Baytril 3 C-X 4/22/2014 Y82 1st Laxative, Baytril 3 C-X 4/23/2014 Y273 1st Suprio 3 C-X 4/24/2014 2811 1st Laxative, Nuflor 3 C-X 4/24/2014 wI 1st Laxative, Nuflor, sulfa 3 C-X 4/24/2014 1100 1st Baytril 3 C-X 4/25/2014 841 1st Laxative 3 C-X 4/25/2014 longhorn 1st Laxative 3 C-X 4/25/2014 452 1st Laxative, Nuflor

* indicates treatment of C-X through the nasal passages (2 cc)

Example 12

Treating Mastitis in Animals

The problem of mastitis in the mammary glands in animals is usually due to infections caused by E. coli or Staphyloccous aureus and other bacterial pathogens. The nipple becomes inflamed and eventually the condition can spread to all other sectors of the breast. Milk production stops. If left untreated, the animal may die. Most antibiotics for treatment are expensive and ineffective. For the past three months, a sheep and two dairy cows suffering from mastitis have been treated with 15 ml of solution per teat. Fig. 25A shows the teat of a sheep with mastitis. The treated animal was well developed in relation to the disease and would not die but remain healthy. The mammary gland has ceased to function. Preparation C-3 was injected using a syringe (FIG. 25B). Two cows suffering from mastitis were in the early stages of the disease. Each was treated with 15 ml per infected nipple and complete recovery was noted within 24 hours.

Exemplary Compositions and Treatment of Mastitis in Farm Animals

Based on 90 ml of water:

5 mg Cremophor or other suitable surfactants

0.7 ml propanoic acid and 0.2 ml isoamyl hexanoate

The composition was shaken well and administered to the cow up to 15 ml per nipple with a syringe. Cremophor acts to bring the ingredients of composition C-3 into solution.

Example 13

Testing MIC C-3 and C-4 compositions

MIC protocol for C-3 and C-4 testing

The cloudy bacterial culture grown in the appropriate nutrient broth was adjusted to OD ₆₅₀ =0.4 and then diluted 1:100 in the broth, which is 1×10 ⁶ cfu/ml. 50 μl of this culture was added to each well, except for the negative control, which was supplemented with 50 μl of broth. The final number of bacteria in each well was 5×10 ⁶ cfu.

20 µl of stock solution of antibiotic B-23 was added to 480 µl of broth. 250 µl of this solution was diluted 1:2. This was repeated twice to prepare four successively diluted antibacterial solutions. The dilutions were such that the final antibiotic concentration in the respective wells was 1%, 0.5%, 0.25%, and 0.125% antibiotic B-23 stock solution.

A 96-well microtiter plate was used. A total of 6 treatments were applied: 1%, 0.5%, 0.25%, 0.125%, 0.061%, 0.03%, and 0% antibiotic with bacterial inoculum; and without bacterial inoculum. Each treatment was repeated three times.

The broth was added to each well to a final volume of 200 μl. In wells without bacterial inoculum or antibiotic solution, an additional 50 µl of broth was added.

The microtiter plates were incubated under suitable growth conditions, and the results were tabulated at time points. Endpoints are selected when the positive control becomes cloudy. The MIC point was taken at the lowest concentration at which growth was not evident.

results

The minimum inhibitory concentrations were as follows for the following microorganisms:

Bacillus subtilis : 0.06125%

Vibrio cholerae : 0.06125%

Pseudomonas aeruginosa : 0.125%

Salmonella enterica serovar Typhimurium: 0.06125%

Escherichia coli : 0.125%

Methicillin-resistant Staphylococcus aureus : 0.06125%

For the other MIC tests, potato dextrose broth was used instead of nutrient broth and tested in the same manner. Results:

Erwinia amylovora : 0.0612%

Lactobacillus sp.: 0.0625%

Erwinia carotovora : 0.125%

The results demonstrate that C-3 and C-4 can be used to treat diseases in plants, animals and humans caused by microorganisms. Such diseases include plant diseases caused by Erwinia and problems in grain fermentation in ethanol production caused by Lactobacillus spp., biofilms formed by Pseudomonas bacteria. Other diseases include foodborne infections caused by Salmonella, E. coli and methicillin-resistant Staphylococcus aureus is the main cause of these diseases.

Example 14

Raspberry processing

The results described in this paper show that C-X technologies can be applied to preserve fruit and vegetables during transport and storage. Preparation C-3 was mixed in the form of two compositions: 1 ml of solution C-3 per 10 g of bentonite (mixture 1:10); 1 ml of C-3 solution with 20 g of bentonite (mixture 1:20) or other carrier. 1 gram of the mixture was placed in a small plastic cup with store-bought raspberries. The materials were placed in a small transparent plastic box, which was sealed and kept at room temperature for 1 week, then examined for the presence of contaminating fungi. The results obtained indicate that the natural microflora of the fruit quickly leads to decay after 1 week at room temperature (Fig. 26A). However, the use of a 1:10 mixture did not result in rot (FIG. 26B). However, the 1:20 mixture did not work as well as the 1:10 mixture, and at least one berry rotted. However, a 1:20 mixture can be used to prevent berry rot and the peeled berries remain edible. A similar experiment was done with store-bought tasty Thompson grapes with similar results, with the control grapes showing rotting while the treated grapes did not. The grapes also remained edible, with 4 people eating them and giving them an acceptable rating.

Example 15

Treatment of food poisoning and/or indigestion in humans using C-X.

The symptoms and characteristics of food poisoning and/or indigestion in humans are similar to those that occur in animals suffering from diarrhea. For example, possible symptoms include: abdominal pain, diarrhea (often bloody), fever and chills, headache, nausea, vomiting, general weakness (may be severe). Most people just suffer (12-48 hours), do their best to rest, do rehydration therapy, replacing the fluid and minerals that are lost due to diarrhea and vomiting. There doesn't seem to be a remedy available that provides instant relief.

However, in ten volunteers suffering from one or more of these symptoms, at least 10-15 ml of 1% C-3 was taken orally at the onset of symptoms or within a few hours of the onset of symptoms. In all cases, patients described improvements within one to two hours after taking the remedy. Fever, abdominal pain, diarrhea and vomiting ceased, and patients recovered completely. All patients were adults, of both sexes, both male and female. One patient, however, reported that he did not notice any difference in the condition of the stomach after taking a 10 ml dose of 1% C-3. Although not wishing to be bound by any particular theory, it is assumed that the patient had a viral infection of the stomach, which is not amenable to C-X. However, the fact that 90% of people treated have an immediate and complete recovery, combined with animal studies of diarrhea, supports the hypothesis that C-3 can be used to treat people suffering from indigestion and poisoning caused by bacteria. . This hypothesis is further supported by the impressive minimum inhibitory concentrations of E coli and S. aureus , which are two known causative agents of food poisoning in humans (Example 13).

Example 16

Mastitis in dairy cattle breeding and agricultural technology

Treatment:

A preparation containing 2% composition C-3 with 5 mg of Cremophor (non-ionic solvent) in pure water was thoroughly mixed and used as a therapeutic agent. Eight dairy cows and bulls suffering from preclinical and subclinical mastitis were treated with 12 ml per teat. In seven cases, the treatment was repeated within one day. In all cases, the animals recovered completely the next day. Although not wishing to be bound by any particular theory, the recovery of the animals is likely due to the fact that the most common bacterial causes of mastitis, such as E. coli and S. aureus , which are highly sensitive to the C-X compositions described in this document (see example 13.)

Example 17

Detergent testing S-3

Several detergents received through a trial order have been tested with C-3 solution for effectiveness on surfaces known to be attacked by various pathogens. These surfaces include the laboratory floor, and the women's bathroom floor, the toilet bowl, and the doorknob. To test the floor, about 5 ml of each of the detergent solutions (1 ml of C-3 in 100 ml of deionized water) was poured onto different areas of the floor and wiped dry with a paper towel. When this area of the floor dried, it was wiped with a Kimwipe cloth and then this cloth was swept over the entire surface of the potato-dextrose broth in Petri dishes. To test the toilet bowl, a paper towel was dampened with a detergent solution and part of the surface was wiped. After the surface dried, Kimwipe was used again, and then edge to edge was swept over the surface of the potato dextrose agar Petri dishes. The procedure for testing the doorknob was the same as for the toilet sink, except for only one of the detergents, along with controls. The results are presented in tables 9 and 10 below.

Table 9: Lab Floor Results

Experiment 1 Experiment 2 Detergent volume Number of colonies Number of colonies Control 22 23 Sucragel CF 1 ml 1 0 Chemoxide CAW 2 ml 1 2 BioSoft D40 0.5 ml 0 3 Lathanol LAL 1 g 2 1 BioTerge AS-40 1 ml 1 2 Nacconol 90G 1 g 1 4 potassium cocoate 2 ml 1 1

Table 9 shows the number of bacterial or fungal colonies grown on potato dextrose broth in petri dishes that were streaked with samples that were wiped with various detergents or only with Kimwipe as a control after 48 hours. One milliliter of C-3 was used per 100 ml of deionized water.

Table 10: Testing Women's Washroom Detergents

Detergent volume Floor covering colonies Toilet sink colonies doorknob colonies Control 12 6 2 Sucragel CF 1 ml 1 0 0 Chemoxide CAW 2 ml 0 1 BioSoft D40 0.5 ml 0 0 Lathanol LAL 1 g 2 16 BioTerge AS-40 1ml 4 21 potassium cocoate 1 g 0 4 Nacconol 90G 2 ml 1 0

Table 10 shows the results of women's restroom testing on various surfaces (floor, toilet, and doorknob), and the number of bacterial or fungal colonies taken from Kimwipe surfaces that have grown after 48 hours in petri dishes in potato dextrose broth.

Example 18

Verticillium experiment

Thirty pea seeds were inoculated with Verticillium sp., placed on a petri dish with a growing fungus. The seeds were rotated freely, then the fungus samples were scraped off and placed with pea seeds in a Petri dish, which was sealed with parafilm and left for three days. Three days later, potato dextrose agar plates were placed in the center of the sterilized caps and filled with 50 µl C-3, 20 µl C-3, or left blank as controls. Ten pea seeds from the inoculated group were placed in each of three Petri dishes containing potato dextrose agar with filled or unfilled caps. Peas were left for two days, and then checked for mushroom growth and germination. The results of the experiment are presented in table 11.

Table 11: Verticillium inoculated pea seeds

Treatment Mushroom Growth Percentage Control 100 20 µl C-3 0 50 µl C-3 0

The percentage of pea seeds infected with Verticillium sp. which germinated and showed mushroom growth after 48 hours in the control variant of the experiment (without C-3), with 20 μl of C-3 and 50 μl of C-3.

Example 19

The gingerbread experiment

Camelina seeds known to be susceptible to various fungal and bacterial pathogens were taken and placed with C-3 to determine if fungal and bacterial growth was likely to stop. Some Petri dishes with potato dextrose broth were used in conjunction with caps to accommodate C-3. About forty seeds were placed in one of the Petri dishes and an empty, sterilized cap was placed in the center as a control group. These Petri dishes were sealed with parafilm and left for two days to determine germination and fungal and bacterial growth. More than a hundred seeds were placed on another Petri dish with sterilized caps filled with 50 µl of C-3. These seeds were left with C-3 in tightly sealed parafilm Petri dishes for the following time intervals: 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours and 48 hours, after which twenty to thirty seeds were taken out and placed separately on a Petri dish. For each of the petri dish intervals, the seeds were kept for 48 hours and then checked for germination and growth of pathogens.

Table 12: Camelina Seed Germination and Pathogenic Growth

Number of seeds per Petri dish Germination, % Percentage with pathogenic growth Control 39 100 56 50 µl C-3: 1 hour 29 97 45 2 hours 25 96 40 4 hours 22 100 9

Table 12 shows the percentage of germination of infected camelina seeds and the percentage with pathogenic growth, which were placed without C-3 (control), or with, covered with 50 μl of C-3 at hourly intervals. All samples were recorded 48 hours after they were placed on Petri dishes with potato dextrose broth.

The description of each patent, patent application, and publication cited herein is incorporated herein by reference in its entirety.

Although the present invention has been described with reference to specific embodiments of the invention, it is obvious that other embodiments of the invention and variations of this invention can be developed by others skilled in the art without departing from the spirit and scope of the present invention. The appended claims are intended to be construed to include all such variations and equivalent embodiments.

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SEQUENCE LIST

<110> Strobel, Gary A.

Blatt, Bryan

<120> COMPOSITIONS OF VOLATILE ORGANIC SUBSTANCES WITH

ANTIMICROBIAL ACTIVITY

<130> 206054-0001-00-WO.603297

<150> US 61/842362

<151> 2013-07-02

<150> US 61/948902

<151> 2014-03-06

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 480

<212> DNA

<213> Fusarium subglutinans

<400> 1

aacataccaa ttgttgcctc ggcggatcag cccgctcccg gtaaaacgggg acggcccgcc

60

agaggacccc taaactctgt ttctatatgt aacttctgag taaaaccata aataaatcaa

120

aactttcaac aacggatctc ttggttctgg catcgatgaa gaacgcagca aaatgcgata

180

agtaatgtga attgcagaat tcagtgaatc atcgaatctt tgaacgcaca ttgcgcccgc

240

cagtattctg gcgggcatgc ctgttcgagc gtcatttcaa ccctcaagcc cagcttggtg

300

ttgggactcg cgagtcaaat cgcgttcccc aaattgattg gcggtcacgt cgagcttcca

360

tagcgtagta gtaaaaccct cgttactggt aatcgtcgcg gccacgccgt taaaccccaa

420

cttctgaatg ttgacctcgg atcaggtagg aatacccgct gaacttaagc atatcaataa

480

<210> 2

<211> 478

<212> DNA

<213> Fusarium subglutinans

<400> 2

cataccaatt gttgcctcgg cggatcagcc cgctcccggt aaaacgggac ggcccgccag

60

aggacccta aactctgttt ctatatgtaa cttctgagta aaaccataaa taaatcaaaa

120

ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcaaa atgcgataag

180

taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt gcgcccgcca

240

gtattctggc gggcatgcct gttcgagcgt catttcaacc ctcaagccca gcttggtgtt

300

gggactcgcg agtcaaatcg cgttccccaa attgattggc ggtcacgtcg agcttccata

360

gcgtagtagt aaaaccctcg ttactggtaa tcgtcgcggc cacgccgtta aaccccaact

420

tctgaatgtt gacctcggat caggtaggaa tacccgctga acttaagcat atcaataa

478

<210> 3

<211> 488

<212> DNA

<213> Fusarium sp

<400> 3

cttaatgttg ccctcggcgga tcagcccgcg ccccgtaaaa cgggacggcc cgccagagga

60

cccaaactct aatgtttctt attgtaactt ctgagtaaaa caaacaaata aatcaaaact

120

ttcaacaacg gatctcttgg ttctggcatc gatgaagaac gcagcaaaat gcgataagta

180

atgtgaattg cagaattcag tgaatcatcg aatctttgaa cgcacattgc gcccgctggt

240

attccggcgg gcatgcctgt tcgagcgtca tttcaaccct caagcccccg ggtttggtgt

300

tggggatcgg ctctgccttc tggcggtgcc gcccccgaaa tacattggcg gtctcgctgc

360

agcctccatt gcgtagtagc taacacctcg caactggaac gcggcgcggc catgccgtaa

420

aaccccaact tctgaatgtt gacctcggat caggtaggaa tacccgctga acttaagcat

480

atcaatag

488

<210> 4

<211> 477

<212> DNA

<213> Fusarium culmorum

<400> 4

cataccttat gttgcctcgg cggatcagcc cgcgccccgt aaaaagggac ggcccgccgc

60

aggaacccta aactctgttt ttagtggaac ttctgagtat aaaaaacaaa taaatcaaaa

120

ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcaaa atgcgataag

180

taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt gcgcccgcca

240

gtattctggc gggcatgcct gttcgagcgt catttcaacc ctcaagccca gcttggtgtt

300

gggagctgca gtcctgctgc actccccaaa tacattggcg gtcacgtcga gcttccatag

360

cgtagtaatt tacatatcgt tactggtaat cgtcgcggcc acgccgttaa accccaactt

420

ctgaatgttg acctcggatc aggtaggaat acccgctgaa cttaagcata tcaatag

477

<210> 5

<211> 485

<212> DNA

<213> Fusarium avenaceum

<400> 5

cagaagttgg ggttttacgg catggccgcg ccgcgttcca gttgcgaggt gttagctact

60

acgcaatgga ggctgcagcg agaccgccaa tgtatttcgg gggcggcacc gccagaaggc

120

agagccgatc cccaacacca aacccgggggg cttgaggggtt gaaatgacgc tcgaacaggc

180

atgcccgccg gaataccagc gggcgcaatg tgcgttcaaa gattcgatga ttcactgaat

240

tctgcaattc acattactta tcgcattttg ctgcgttctt catcgatgcc agaaccaaga

300

gatccgttgt tgaaagtttt gatttatttg tttgttttac tcagaagtta caataagaaa

360

cattagagtt tgggtcctct ggcgggccgt cccgttttac ggggcgcggg ctgatccgcc

420

gaggcaacat taaggtatgt tcacaggggt ttgggagttg taaactcggt aatgatccct

480

ccgca

485

<210> 6

<211> 469

<212> DNA

<213> Fusarium subglutinans

<400> 6

cagaagttgg ggtttaacgg cgtggccgcg acgattacca gtaacgaggg ttttactactact

60

acgctatgga agctcgacgt gaccgccaat caatttgggg aacgcgattt gactcgcgag

120

tcccaacacc aagctgggct tgaggggttga aatgacgctc gaacaggcat gcccgccaga

180

atactggcgg gcgcaatgtg cgttcaaaga ttcgatgatt cactgaattc tgcaattcac

240

attacttatc gcattttgct gcgttcttca tcgatgccag aaccaagaga tccgttgttg

300

aaagttttga tttatttatg gttttactca gaagttacat atagaaacag agtttaggggg

360

tcctctggcg ggccgtcccg ttttaccgggg agcgggctga tccgccgagg caacaattgg

420

tatgttcaca ggggtttgggg agttgtaaac tcggtaatga tccctccgc

469

<210> 7

<211> 480

<212> DNA

<213> Fusarium avenaceum

<400> 7

gaagttgggg ttttacggca tggccgcgcc gcgttccagt tgcgaggtgt tagctactac

60

gcaatggagg ctgcagcgag accgccaatg tatttcgggg gcggcaccgc cagaaggcag

120

agccgatccc caacaccaaa cccgggggct tgaggggttga aatgacgctc gaacaggcat

180

gcccgccgga ataccagcgg gcgcaatgtg cgttcaaaga ttcgatgatt cactgaattc

240

tgcaattcac attacttatc gcattttgct gcgttcttca tcgatgccag aaccaagaga

300

tccgttgttg aaagttttga tttatttgtt tgttttactc agaagttaca ataagaaaca

360

ttagagtttg ggtcctctgg cgggccgtcc cgttttacgg ggcgcgggct gatccgccga

420

ggcaacatta aggtatgttc acaggggttt gggagttgta aactcggtaa tgatccctcc

480

<210> 8

<211> 468

<212> DNA

<213> Fusarium subglutinans

<400> 8

gaagttgggg tttaacggcg tggccgcgac gattaccagt aacgaggggtt ttactactac

60

gctatggaag ctcgacgtga ccgccaatca atttggggaa cgcgatttga ctcgcgagtc

120

ccaacaccaa gctgggcttg agggttgaaa tgacgctcga acaggcatgc ccgccagaat

180

actggcgggc gcaatgtgcg ttcaaagatt cgatgattca ctgaattctg caattcacat

240

tacttatcgc attttgctgc gttcttcatc gatgccagaa ccaagagatc cgttgttgaa

300

agttttgatt tatttatggt tttactcaga agttacatat agaaacagag tttagggggtc

360

ctctggcggg ccgtcccgtt ttaccgggag cgggctgatc cgccgaggca acaattggta

420

tgttcacagg ggttggggag ttgtaaactc ggtaatgatc cctccgca

468

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Chemically synthesized

<400> 9

tccgtaggtg aacctgcgg

19

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Chemically synthesized

<400> 10

tcctccgctt attgatatgc

20

<---

Claims (26)

1. An antimicrobial composition suitable for use directly or as a component in other compositions and having antimicrobial activity, containing a combination of isoamylhexanoate and at least one acid selected from the group consisting of propanoic acid and isobutyric acid, and an acceptable carrier. 2. An antimicrobial composition according to claim 1, formulated so that it can be used as a human food product or a product comprising it. 3. The antimicrobial composition of claim. 1, formulated so that it can be used as animal feed or feed comprising it. 4. An antimicrobial formulation according to claim 1 formulated to be useful as a human nutritional supplement or a nutritional supplement comprising it. 5. An antimicrobial formulation according to claim 1 formulated to be used as a nutritional supplement for an animal or a nutritional supplement comprising it. 6. The antimicrobial composition of claim 1 formulated to be useful as a human food supplement or a food supplement comprising it. 7. An antimicrobial composition according to claim 1, formulated so that it can be used as a feed additive for an animal or a feed additive comprising it. 8. An antimicrobial composition according to claim 1, formulated so that it can be used as a dietary supplement for a person or a dietary supplement including it. 9. An antimicrobial composition according to claim 1, formulated so that it can be used as a dietary supplement for an animal or a dietary supplement including it. 10. Antimicrobial composition for the treatment of human or animal excrement, containing the antimicrobial composition according to claim 1. 11. Composition containing an antimicrobial composition with antimicrobial activity, and at least one other component, where the antimicrobial composition contains: (i) a combination of isoamylhexanoate and at least one acid selected from the group consisting of propanoic acid and isobutyric acid, and (ii) an acceptable carrier. 12. Cleaning composition for reducing the growth of microorganisms on surfaces or tools or planes, containing an antimicrobial composition according to claim 11. 13. The cleaning composition of claim 12, wherein the surfaces, tools, or planes are selected from medical instruments, agricultural plants, and livestock implements. 14. Cleaning composition according to claim 12, suitable for use as a solution or aerosol for treating surfaces, tools or planes in one or more of the following: hospitals; homes or restaurants or food preparation facilities; industrial centers of the food industry; and other places where food is processed, stored, transported or sold and where bacterial contamination may be a problem. 15. Cleaning composition for decontamination, decomposition or deodorization of human or animal feces, containing an antimicrobial composition according to claim 1. 16. A cleaning composition for treating faeces in one or more places selected from the following: a latrine, a cat litter box, an animal stall, a barn, a chicken coop, a pig shed, a pet's place in the house, and a zoo, containing an antimicrobial composition according to claim 1. 17. A cleaning composition for treating faeces in one or more places selected from the following: a latrine, a cat litter box, an animal stall, a barn, a chicken coop, a pig shed, a pet's place in the house, and a zoo, containing an antimicrobial composition according to claim 11. 18. Pharmaceutical composition for the treatment of a human or animal having a microbial infection, containing an antimicrobial composition according to claim 1. 19. A pharmaceutical composition for the treatment of a human or animal having a microbial infection, comprising a composition according to claim 11. 20. A method of treating a human or animal having a microbial infection, comprising administering to the human or animal an effective amount of the antimicrobial composition of claim 1. 21. A method of treating a human or animal having a microbial infection, comprising administering to the human or animal an effective amount of the antimicrobial composition of claim 11. 22. The method of claim 20, wherein the microbial infection is caused by Escherichia coli , Staphylococcus aureus , or Salmonella. 23. The method of claim 21, wherein the microbial infection is caused by Escherichia coli , Staphylococcus aureus , or Salmonella. 24. The method of claim 21, wherein the at least one acid in combination is propanoic acid and isobutyric acid in a combination of about 1:1 by volume.

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