HK1170634B - Antimicrobial compositions and related methods of use - Google Patents
Antimicrobial compositions and related methods of use Download PDFInfo
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Description
The present application claims priority from application serial No.61/214,752 filed on 27/4/2009, application serial No.61/257,319 filed on 11/2/2009, and application serial No.61/315,611 filed on 19/3/2010, each of which is incorporated herein by reference in its entirety.
Background
The identification and development of biocides for controlling various mold and plant diseases, etc. has made great progress. However, most commercial biocides and pesticides in use are compounds classified as carcinogens or toxic to wild animals and plants and other non-target species. For example, methyl bromide is widely used as a soil fumigant and treatment of post-harvest microbial infections. Toxic to humans and harmful to the environment will eventually result in the cessation of methyl bromide and other various synthetic biocides/pesticides. Thus, current work has focused on identifying and developing biomimetic compositions that are natural or that exhibit similar antimicrobial or insecticidal effects.
One such method involves endophytes (endophytes) and associated volatile byproducts. Endophytes are defined in the art as microorganisms that reside in the interstitial spaces of living plant tissue, but are not generally considered parasitic. In particular, considerable interest has arisen in endophytes found in connection with rain forest plants due to the antibacterial properties of their volatile by-products. It has been found that various members of the genus Muscodor (i.e., m.albus, m.roseus, and m.vitigenus) produce volatile by-products that exhibit antibacterial or insecticidal properties. However, representative by-products of each species include various naphthalene and/or azulene (azulene) derivatives. Such compounds, along with other byproduct components, may be toxic or unhealthy, and the corresponding mixtures are considered unsuitable for a variety of end-use applications. Thus, the art continues to search for natural compositions and to develop biomimetic compositions free of the above compounds that are safe for human use and that exhibit effective antimicrobial properties.
Disclosure of Invention
With reference to the above, it is an object of the present invention to provide seasonings (flavors) with antimicrobial compositions and/or methods of use thereof that overcome various deficiencies and inadequacies of the prior art, including those described above. It will be appreciated by those skilled in the art that one or more aspects of the present invention may serve certain purposes, while one or other aspects may serve certain other purposes. It is not possible for each object to apply equally each aspect of the invention in all its aspects. Accordingly, reference may be made to the following objects in lieu of any one aspect of the present invention.
It is an object of the present invention to provide Muscodor species (Muscodor species) and their volatile by-products, which are free of compounds related to naphthalene and azulene (non-GRAS compounds), and to provide methods for preventing, inhibiting and/or eradicating microbial infections.
It is another object of the present invention to provide a system for combating microbial infections, said system comprising this species or a strain thereof and associated volatile by-products, and a non-indigenous (non-indigenous) medium or substrate (substrate).
It is another object of the present invention to provide such systems and/or related methods of use for, but not limited to, human and animal food, products (products), plants, plant parts, seeds, agricultural crops and other organic materials, packaging materials, building materials, fibers, textiles, clothing and pharmaceutical and/or medical applications.
It is another object of the present invention to provide a series of biomimetic artificial compositions that exhibit similar antimicrobial activity to such Muscodor species, either instead of or in combination therewith.
It is an object of the present invention to provide one or more compositions of ingredients that are edible or safe for human use and use.
It is an object of the present invention to provide a system, composite or article (articule) comprising such a non-natural, biomimetic composition in combination with a medium or matrix to prevent, inhibit and/or eradicate microbial infection. It is another object of the present invention to provide such systems, composites and/or articles for use in applications such as those described above or elsewhere herein.
It is also an object of the present invention to provide a method for antimicrobial and/or insecticidal treatment comprising such a composition, without limitation to the medium, carrier or matrix.
Other objects, features, benefits and advantages of the present invention will be apparent from the summary of the invention and the description of the specific embodiments below, and will be readily apparent to those skilled in the art having knowledge of a variety of antimicrobial compositions and related treatments. Such objects, features, benefits and advantages will be apparent from a consideration of the attached examples, data and drawings, and all reasonable inferences thereof, alone or in combination with the references incorporated herein.
In one aspect, the invention can relate to a system comprising at least one m.crispans strain, a volatile byproduct thereof, or a vapor of such a volatile byproduct and an extrinsic medium or matrix. Such media or matrices may be as described herein, or as understood by one of skill in the art. In any event, the strain may be provided in the form of a biologically pure culture, optionally in combination with carrier components suitable for use in media/substrate contact or end use applications, such culture being sufficiently viable to produce volatile byproducts. Crispans by-products or by-product modifications or their corresponding vapors in accordance with the present invention are as described elsewhere herein in composition.
Accordingly, the present invention also relates to the use of such systems and/or their volatile fungal byproducts to provide antimicrobial action. Such methods may include providing an extrinsic substrate or medium capable of supporting microbial activity or growth; and contacting the substrate or medium with a culture of the m.crispans strain, volatile by-products thereof and/or vapours of such by-products. In certain embodiments, such contacting may comprise contacting the strain on, near, or adjacent to the medium or substrate. In certain other embodiments, byproducts or byproduct modifications of m.crispans or their corresponding vapors may be injected into or contacted with such media or substrates.
Without being limited by any such system or method, such substrates may be selected from: food or its products, packaging components for food and other perishable items, fibers, fabrics or clothing items, building or construction components, plants, plant surfaces, soil, garbage or waste. Such contact may be biologically active against the presence of microorganisms and/or may be prophylactic (pathogenic).
In one aspect, the invention can relate to non-naturally occurring antimicrobial compositions, whether the components thereof are of natural origin, chemically synthesized, or a combination thereof. Such compositions may comprise compounds selected from the group consisting of alcohol, aldehyde, ketone, acid and/or acid ester components of biomimetic Muscodor sp byproduct compositions, which may be free of fused ring aromatics, substituted fused ring aromatics, and hydrogenated derivatives of such compounds (hydro derivitives). In certain non-limiting embodiments, the composition can comprise an acid component selected from the group consisting of acetic acid, isobutyric acid, propionic acid, and combinations thereof.
In certain embodiments, the present invention may relate to an antimicrobial composition of natural origin comprising C2To about C5The acid component of (a); c2To about C5An ester component of (a); and at least two C's isolatable from volatile by-products of isolated cultures of Muscodor crispans2To about C5The composition may have a pathogen activity profile different from that of the isolated cultured Muscodor sp. Such acid components may be selected from isobutyric acid, propionic acid, and combinations thereof.Independently, such ester component may be selected from C4Acetic acid ester, C5Acetates and combinations thereof.
In certain embodiments, the composition may comprise from about 8 to about 10 components that can be isolated from volatile byproducts of m. In certain such embodiments, each component of the composition can be isolated from volatile byproducts. Since the composition may be derived from nature, each such component may be a fermentation product, and the fermentation may be selected from bacterial, yeast and/or fungal fermentations. In any event, each such component of the composition is generally considered safe for human use in accordance with chapter 21 of the U.S. Federal State Code of Federal Regulations and the associated chapters and/or Regulations.
Regardless, in certain non-limiting embodiments, such a separable component can be isobutyric acid. In certain such embodiments, at least a portion of the isobutyric acid may be replaced by propionic acid. In such or other non-limiting embodiments, such a separable component can be 2-butanone. In certain such embodiments, at least a portion of the 2-butanone may be replaced by acetic acid, propionic acid, or a combination thereof. In such or other non-limiting embodiments, such a separable component can be ethanol. In certain such embodiments, at least a portion of the ethanol may be replaced by acetic acid. Such naturally derived compositions may comprise a surfactant component, regardless of the identity or amount of any such acid component, ester component, and/or isolatable component. In certain such embodiments, a biosurfactant may be added thereto. The biosurfactant may be a rhamnolipid component selected from the group consisting of mono-rhamnolipids, bis-rhamnolipids and combinations thereof, but is not limited thereto.
Alternatively, the present invention may also relate to synthetic, non-naturally derived antimicrobial compositions. Such compositions may comprise C2To about C5The acid component of (a); c2To about C5An ester component of (a); and at least two C's isolatable from volatile by-products of isolated cultures of Muscodor crispans2To about C5Composition, etiology of said compositionThe activity profile may be different from the pathogen activity profile of the isolated cultured Muscodor sp. Such acids, esters and/or separable components may be as described above or as described elsewhere herein. Regardless, the antimicrobial composition can include a surfactant component. In certain such non-limiting embodiments, the surfactant can be a rhamnolipid component selected from the group consisting of mono-rhamnolipids, bis-rhamnolipids, and combinations thereof.
In one aspect, the present invention can relate to a biomimetic antimicrobial composition comprising a compound selected from C2To about C5And combinations and subcombinations thereof, which composition is not isolated from a Muscodor sp. As described elsewhere herein, such liquid mixtures may be volatile at room and/or ambient temperature. As will be understood by those skilled in the art, in connection with such compositions and compounds thereof, the term "about" may refer to carbon and/or methylene homologues having the corresponding molecular weight and/or structural isomers limited only by the volatility of one or more of the other components, mixtures of compounds, and at least a portion of the room/ambient temperature of the resulting composition. In certain non-limiting embodiments, the composition can comprise alcohol, aldehyde, ketone, acid, and acid ester compounds selected from those components that biosimilar m. Such compositions can include chemically synthesized compounds, compounds isolated from bacterial fermentation, and combinations of such compounds. In certain non-limiting embodiments, the composition can comprise an acid component selected from the group consisting of acetic acid, isobutyric acid, propionic acid, and combinations thereof.
In one aspect, the invention may also relate to non-naturally occurring, whether naturally derived and/or chemically synthesized, antimicrobial compositions comprising a compound selected from C2To about C5The selected compounds are generally considered to be safe for human use ("GRAS"), a designation provided in chapter 21 of the federal regulations and associated chapters and/or regulations in the united states. In certain non-limiting embodiments, this is doneThe compounds may be selected from the alcohol, ketone, acid and/or acid ester components that biologically mimic the m. In certain embodiments, the microbial activity/mortality profile thereof is different from the microbial activity/mortality profile of m.crispans or m.albus, volatile byproducts thereof, and/or corresponding synthetic byproduct compositions thereof. Regardless, in certain such embodiments, the composition can comprise an acid component selected from the group consisting of acetic acid, isobutyric acid, propionic acid, and combinations thereof.
In one aspect, the invention can include a composition comprising a composition of the invention, a surfactant component; such surfactant components are separate or may be added to the carrier component. In certain embodiments, the surfactant can be a biosurfactant, and such biosurfactant can be a rhamnolipid component selected from the group consisting of mono-rhamnolipids, bis-rhamnolipids, and combinations thereof.
In one aspect, the invention can also relate to a system or composite comprising a composition of the invention and a matrix or media component. Such compositions may be as described above or as described elsewhere herein. The matrix may be selected from: food or its products, packaging components (e.g., films or packaging materials) for food and other perishable items, fibers, fabrics or clothing items, building or construction components, human tissue, plants, plant surfaces, soil, trash or waste, but are not limited thereto. In certain embodiments, such compositions, whether liquid or gaseous, may be added to or contacted with such media, substrates, or substrate surfaces.
Thus, the present invention may also relate to a method of treating, preventing, inhibiting, eliminating and/or affecting the activity of a microorganism or an insect (insect). Such methods may include: the compositions of the present invention, including but not limited to one or more of the compositions described herein, are provided and the microorganism or insect or article/substrate capable of supporting microorganism or insect activity is contacted with such compositions in an amount at least sufficient to partially affect the activity of the microorganism or insect. Such microorganisms (e.g., fungi, bacteria, or viruses) or insects may be in, on, or near the surface of the substrate in those media described above. Thus, such contacting may be direct contacting and/or contacting upon volatilization of such composition. Regardless, such treatments may be active against the presence of microorganisms or insects and/or disease preventative. As indicated elsewhere herein, treatment may be considered to be in the field of microbial or insect death and/or inhibition of growth or activity.
According to certain embodiments of the present invention, compositions comprising certain Food and Flavor Compounds (FFCs) are inhibitory and/or lethal, particularly to certain pathogenic fungi, bacteria, and other microorganisms of agricultural, pharmaceutical, or commercial or industrial aspects. Such compositions can be distinguished from any existing mixture comprising compounds of biological origin: for example, the compositions of the present invention do not contain any naphthalene or azulene (non-GRAS compounds) derived material. Rather, such compositions may comprise a mixture of organic compounds, each of which is considered a food or flavor (i.e., GRAS).
The present invention demonstrates the properties of such compositions, their preparation and application to a variety of articles such as, but not limited to, food, fiber, appliances and building surfaces to preserve their integrity and protect them from various fungi (mold and other microorganisms). Such compositions may also find application in the preservation of building structures, plant parts, and even clothing products. Furthermore, as demonstrated below, such compositions can have an adverse effect on Mycobacterium tuberculosis (Mycobacterium tuberculosis) that causes tuberculosis, including at least three drug resistant strains.
Drawings
FIG. 1: photographs illustrating the killing effect of FFC against drug-resistant clinical cultures of mycobacterium tuberculosis after 2 days of exposure.
FIG. 2: a series of photographs illustrating the prevention of fungal growth (mildew) on cheese by a number of methods employing FFC.
FIG. 3: illustrating the protective effect of FFC on yam after 2 days of storage in the presence of 0.2ml of FFC composition. The yam was photographed 10 days later (test on the left side; control on the right side).
FIG. 4: protection of FFC from spoilage of garbage kept at 30 ℃ for 10 days.
FIG. 5: effects against tomato rot/wilt were demonstrated with a control plate of clavibacter michiganense (Cmichiganense) on the left and a plate treated with 20 microliters of the FFC composition of the invention on the right.
FIG. 6: the effect of the FFC composition of the present invention added to the skin cream product (skin cream product) was confirmed.
Figures 7A-B and 8 illustrate the structure of various non-limiting, representative mono and di-rhamnolipid compounds, according to certain non-limiting embodiments of the present invention.
FIG. 9: according to certain non-limiting embodiments of the present invention, two embodiments of rhamnolipid components are provided, designated R1 and R2, respectively, based on mono and di rhamnolipid structures, which may be used alone or in combination with each other as described in the following examples.
Detailed Description
As described in various non-limiting embodiments, the present invention relates to the use of new Muscodor species and/or their volatile byproducts, as well as non-natural, laboratory-prepared biomimetic compositions comprising common food and flavor compounds that, when added to various media, applied to a surface, or added to an environmental atmosphere (atmosphere), space or volume, bring about a decontamination effect (decontamination) of the desired surface media or volume of undesirable, harmful and/or pathogenic microorganisms, including plant fungi and pathogenic agents of tuberculosis (domestic agents). The invention has extremely important significance and application to modern agriculture, human medicine, food science and industry. It is not obvious that the compositions of the present invention have antimicrobial properties, since any of the individual components are not biologically active per se. The synergistic combination of the component ingredients showed full potential antimicrobial activity.
With respect to the use of such Muscodor species, volatile byproducts thereof, or non-naturally occurring biomimetic compositions comprising FFCs, the contacting may be direct contacting or by exposure to associated vapors with such species, byproducts of the biomimetic composition. As described below, in certain embodiments, while vapor contact can inhibit growth, direct contact with microorganisms may be required to cause bacteria or fungi to die.
Regardless of the mode of contact, the compositions of the present invention may be prepared in the laboratory and comprise chemically synthesized components, components of natural origin, or a combination of such synthetic and natural components. In any event, the composition can mimic the biological effects of the Muscodor byproduct on specific bacterial and fungal species. Alternatively, such compositions may exhibit altered or enhanced antimicrobial activity as compared to the Muscodor fungal byproduct by virtue of the relative concentrations or selection of any one or more of the FFC components thereof.
In certain such embodiments, such compositions may be located on or applied to a substrate or medium, wherein the substrate or medium comprises a protein or cellulosic component that may, is capable of, or does support the growth of a microorganism. Certain embodiments may comprise plants, plant components (e.g., roots, stems, leaves or leaves (leaves) and fruits (products), etc.), and any primary (germination) shoots or seeds, but are not limited thereto. In particular, the composition may be on any plant product, whether it be a fruit, vegetable, tuber (tuber), flower, seed or nut, whether it be pre-harvest or post-harvest, but is not so limited. In the art, certain such plants and/or their products are referred to individually or collectively as crops. Thus, in certain embodiments, the compositions of the present invention may be located on or applied to such crops during any time during development, pre-harvest and/or post-harvest. Likewise, the compositions of the present invention may be applied to or incorporated into beverages, food (e.g., human, pet, and/or animal) products or articles of manufacture that are capable of or do support the growth of microorganisms.
In certain other embodiments of the present invention, such compositions may be located on or applied to a substrate or surface that supports or can support the growth of microorganisms (e.g., yeast and/or fungi, bacteria, and/or viruses). Thus, such substrates or surfaces may comprise any material that may, is capable of, or does support the growth of microorganisms. Such substrates include, but are not limited to, wood, pottery, porcelain, stone, gypsum, drywall, cement, fabric, leather, plastic, and the like.
In certain other embodiments, various compositions of the present invention may be located on, contacted with, or applied to a substrate or surface comprising mammalian or human tissue in the field of pharmaceutical or personal care or hygiene formulations for treating or preventing microbial growth or infection, including but not limited to, nails, hair, teeth or oral cavity, skin and other cellular materials. Exemplary compositions that may be applied, at least in part, to one or more of the other embodiments are described below.
Endophytic fungi were collected from the inside of the tissue of a wild pineapple plant (mini pineapples) growing in the bauhinia amazonii region. Finally, it was shown to produce a mixture of volatile compounds with antimicrobial activity. Using molecular techniques, the fungus was found to have a sequence similar to a member of the genus Muscodor. These fungi are known to produce volatile organic compounds that can act as antimicrobial agents effective against human and plant pathogens. Members of the Muscodor species have been identified using methods such as evolutionary charting (phenotypic characterization) using 18S rDNA and ITS-5.8S rDNA sequence analysis. The sequences to be found in the fungi of the invention and other Muscodorspp. were searched in GenBank using BLAST and compared with other fungi (Bruns et al, 1991; Reynolds and tea 1993; Mitchell et al, 1995; Guarro et al, 1999; Taylor et al, 1999). These isolates were finally determined to be associated with Xylaria (Xylaria) (Worapong et al, 2001a & b). All isolated taxa belonging to Muscodor have similar characteristics, such as relatively slow growth, felt-like mycelium, production of volatile compounds with biological activity, and not harmful to the plants in which they originally resided. Finally, they each share very similar rDNA sequences (Ezra et al, 2004).
Although the fungi of the invention have all the same common characteristics as described above, they also differ from the classification in several respects, thereby distinguishing them from all other Muscodor species and isolates. As more fully illustrated in the examples below, these unique characteristics support the fungus of the present invention as a new species. The new endophytic fungus is proposed to be named Muscodor crispans.
By GC/MS analysis, isolated fungi produced alcohols, esters and low molecular weight acids in the gas phase when grown on Potato Dextrose Agar (PDA). As shown in table 1 below, such compounds include propionic acid, 2-methyl; 1-butanol, 3-methyl, acetate; 1-butanol and ethanol. This organism produced no naphthalene or azulene derivatives (non-GRAS compounds) when grown on PDA, thus distinguishing it from all other Muscodor species currently studied (Muscodor spp.). The odor produced by the fungus became apparent after about 1 week and appeared to increase over time for at least 3 weeks. As shown below, the volatiles of this fungus have inhibitory and lethal biological activity on a variety of plants and human pathogens using standard bioanalytical techniques (Strobel et al, 2001).
Table 1.
| Retention time (minutes) | Compound (I) | MW |
| 2:05 | Acetaldehyde | 44.03 |
| 3:40 | Ethyl acetate | 88.05 |
| 3:51 | 2-butanone | 72.06 |
| 4:08 | Propionic acid, 2-methyl-, methyl ester | 102.07 |
| 4:18 | Ethanol | 46.04 |
| 5:29 | Acetic acid, 2-methylpropyl ester | 116.08 |
| 6:39 | Propionic acid, 2-methyl-, 2-methylpropyl ester | 144.12 |
| 6:46 | 1-propanol, 2-methyl- | 74.07 |
| 6:52 | 2-butenal, 2-methyl-, (E) - | 84.06 |
| 7:12 | 1-Butanol, 3-methyl-, acetate | 130.10 |
| 8:18 | Hexane, 2, 3-dimethyl- | 114.14 |
| 8:21 | Propionic acid, 2-methyl-, 2-methylbutyl ester | 158.13 |
| 8:31 | 1-butanol, 3-methyl- | 88.09 |
| 13:37 | Propionic acid, 2-methyl- | 88.05 |
| 14:41 | Formamide, N- (1-methylpropyl) - | 101.08 |
| 16:44 | Acetic acid, 2-Phenylethyl ester | 164.08 |
| 20:44 | Cyclohexane, 1, 2-dimethyl-3, 5-bis (1-methylethenyl) - | 192.19 |
As noted above, the present invention includes the use of m.crispans and/or volatile by-products thereof in combination with extrinsic media, matrices and/or volumes for antimicrobial action. Such uses and/or applications may be as described herein, or as understood by those skilled in the art, including but not limited to those described in U.S. patent No.6,911,338, which is incorporated herein by reference in its entirety.
Alternatively, as demonstrated by one or more embodiments, a variety of natural and synthetic biomimetic compositions with similar or enhanced effects may also be used to provide results that have not heretofore been obtained through the use of fungi or their volatile byproducts. Unlike the prior art and the by-products of m.crispans, such antimicrobial compositions may contain food and flavor compounds that are generally considered safe for human use and use. A number of representative, non-limiting biomimetic compositions are provided in tables 2-7 below. Various other compositions may comprise a compound selected from any one or more of tables 2-7. Alternatively, any such composition may comprise a compound component in addition to or in place of any of the compounds listed to enhance volatility or improve any other end use or performance property. In certain such compositions, such substituted or added compounds may have GRAS designations and/or be named according to their use at various stages. Alternatively, such compositions can also comprise components found in a volatile by-product of m.crispans and/or components not present in a volatile by-product of another Muscodor sp.
Each such compound may be provided in an effective concentration or percentage range and is commercially available or may be prepared by one skilled in the art. For the latter, fermentation techniques may be used to prepare and isolate such compounds in a natural manner. Alternatively, such compounds may be synthesized chemically. With respect to various non-limiting embodiments of the present invention, each of the compounds of tables 2-7 is available as a fermentation product, and such products and corresponding compositions are available from Jeneil Biotech, Inc, of Saukville, Wisconsin under the trademark Flavorzon.
Table 2: the biomimetic composition of the present invention comprises:
| compound (I) |
| Acetaldehyde |
| Ethyl acetate |
| 2-butanone |
| Propionic acid, 2-methyl-, methyl ester |
| Ethanol |
| Acetic acid, 2-methylpropyl ester |
| Propionic acid, 2-methyl-, 2-methylpropyl ester |
| 1-propanol, 2-methyl- |
| 1-Butanol, 3-methyl-, acetate |
| Propionic acid, 2-methyl-, 2-methylbutyl ester |
| 1-butanol, 3-methyl- |
| Propionic acid |
| Acetic acid, 2-Phenylethyl ester |
Table 3. a biomimetic composition of the present invention comprising:
| compound (I) |
| Acetaldehyde |
| Ethyl acetate |
| 2-butanone |
| Propionic acid, 2-methyl-, methyl ester |
| Ethanol |
| Acetic acid, 2-methylpropyl ester |
| Propionic acid, 2-methyl-, 2-methylpropyl ester |
| 1-propanol, 2-methyl- |
| 1-Butanol, 3-methyl-, acetate |
| Propionic acid, 2-methyl-, 2-methylbutyl ester |
| 1-butanol, 3-methyl- |
| Propionic acid, 2-methyl- |
| Acetic acid, 2-Phenylethyl ester |
| Propionic acid |
Table 4. a biomimetic composition of the present invention comprising:
| compound (I) |
| Acetaldehyde |
| Ethyl acetate |
| 2-butanone |
| Propionic acid, 2-methyl-, methyl ester |
| Acetic acid |
| Acetic acid, 2-methylpropyl ester |
| Propionic acid, 2-methyl-, 2-methylpropyl ester |
| 1-propanol, 2-methyl- |
| 1-Butanol, 3-methyl-, acetate |
| Propionic acid, 2-methyl-, 2-methylbutyl ester |
| 1-butanol, 3-methyl- |
| Propionic acid, 2-methyl- |
| Acetic acid, 2-Phenylethyl ester |
Table 5. a biomimetic composition of the present invention comprising:
| compound (I) |
| Acetaldehyde |
| Ethyl acetate |
| Acetic acid |
| Propionic acid, 2-methyl-, methyl ester |
| Ethanol |
| Acetic acid, 2-methylpropyl ester |
| Propionic acid, 2-methyl-, 2-methylpropyl ester |
| 1-propanol, 2-methyl- |
| 1-Butanol, 3-methyl-, acetate |
| Propionic acid, 2-methyl-,2-Methylbutyl ester |
| 1-butanol, 3-methyl- |
| Propionic acid, 2-methyl- |
| Acetic acid, 2-phenylethyl ester |
Table 6. a biomimetic composition of the present invention comprising:
| compound (I) |
| Acetaldehyde |
| Ethyl acetate |
| Propionic acid |
| Propionic acid, 2-methyl-, methyl ester |
| Ethanol |
| Acetic acid, 2-methylpropyl ester |
| Propionic acid, 2-methyl-, 2-methylpropyl ester |
| 1-propanol, 2-methyl- |
| 1-Butanol, 3-methyl-, acetate |
| Propionic acid, 2-methyl-, 2-methylbutyl ester |
| 1-butanol, 3-methyl- |
| Propionic acid, 2-methyl- |
| Acetic acid, 2-Phenylethyl ester |
Table 7. biomimetic compositions of the present invention comprising various combinations of compounds selected from or comprising the following compounds:
| ％ | compound (I) |
| About 0.1 to about 10 | Acetaldehyde |
| About 0.5 to about 25 | Ethyl acetate |
| About 0.1 to about 15 | 2-butanone |
| About 4 to about 99 | Propionic acid, 2-methyl-, methyl ester |
| About 1.5 to about 40 | Ethanol |
| About 0.1 to about 10 | Acetic acid, 2-methylpropyl ester |
| About 0.1 to about 15 | Propionic acid, 2-methyl-, 2-methylpropyl ester |
| About 0.1 to about 10 | 1-propanol, 2-methyl- |
| About 0.5 to about 25 | 1-Butanol, 3-methyl-, acetate |
| About 0.5 to about 25 | Propionic acid, 2-methyl-, 2-methylbutyl ester |
| About 2 to about 50 | 1-butanol, 3-methyl- |
| About 10 to about 99 | Propionic acid, 2-methyl- |
| About 0.1 to about 10 | Acetic acid, 2-Phenylethyl ester |
With respect to any FFC composition of the present invention, it is contemplated that any compound component thereof, including any compound component cited or inferred herein (such as, but not limited to, any of the components in tables 1-7 and 10 and structural isomers and/or carbon and methylene homologs thereof) can be present in an amount or range independent of any other constituent component. Thus, according to incremental variation (incremental variation), each such compound component may be present in an amount or range of about 0.1 wt.%, (or less), about 0.2 wt.%, about 0.3 wt.%, or about 0.4 wt.%, or/to about 1.0 wt.%, about 1.1 wt.%, about 1.2 wt.%, about 1.3 wt.%, or about 1.4 wt.%, or/to about 2.0 wt.%, about 2.1 wt.%, about 2.2 wt.%, about 2.3 wt.%, or about 2.4 wt.%, or/to about 3.0 wt.%, about 3.1 wt.%, about 3.2 wt.%, about 3.3 wt.%, or about 3.4 wt.%, or/to about 4.0 wt.%, about 4.1 wt.%, about 4.4 wt.%, about 4.1 wt.%, about 4.5 wt.%, about 4 wt.%, or about 4.5 wt.%, about 1.5 wt.%, or about 1.5.0 wt.%, about 1.0 wt.%, or about 1.5.5 wt.%, about 1.0 wt.%, or about 1.5.5.6 wt.%, or about 1.0 wt.%, or about 1.5.0 wt.%, about 6.2 wt.%, about 6.3 wt.%, or about 6.4 wt.%, or about 7.4 wt.%, or about 8.0 wt.%, about 8.1 wt.%, about 8.2 wt.%, about 8.3 wt.%, or about 8.4 wt.%, or about 9.0 wt.%, about 9.1 wt.%, about 9.2 wt.%, about 9.3 wt.%, or about 9.4 wt.%, or about 10.0 wt.%; and, or/to about 10.1wt. -%, or/to about 20.0 wt.%; according to an incremental change, or/to about 20.1wt. -%, or/to about 30.0 wt.%; according to an incremental change, or/to about 30.1wt. -%, or/to about 40.0 wt.%; according to an incremental change, or/to about 40.1wt. -%, or/to about 50.0 wt.%; according to an incremental change, or/to about 50.1wt. -%, or/to about 60.0 wt.%; according to an incremental change, or/to about 60.1wt. -%, or/to about 70.0 wt.%; according to an incremental change, or/to about 70.1wt. -%, or/to about 80.0 wt.%; according to an incremental change, or/to about 80.1wt. -%, or/to about 90.0 wt.%; according to an incremental change, or/to about 90.1wt. -%, or/to about 99.9 wt.% (or more), but not limited thereto. Likewise, regardless of the identity or amount of any particular compound component or combination, any composition of the present invention may be present in an incrementally varying amount (wt.%) or wt.% range, as described above, with 0.1 wt.% to 99.9 wt.% of any composition or medium (e.g., in about 0.1 wt.% to about 1.0 wt.%, about 2.0 wt.%, about 4.0 wt.%, or to about 10.0 wt.%) being added to or applied to an article or substrate, but not limited to.
Unless otherwise indicated, in all cases, the amounts, concentrations, or amounts of a component or ingredient, properties such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as modified by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Finally, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques, and not by way of intending to limit the application of the doctrine of equivalents to the scope of the claims.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth and the examples are reported as precisely as possible. Any numerical value, however, may inherently contain certain errors resulting from the standard deviation found in their respective testing measurements.
The compositions and methods of the present invention may suitably comprise, consist essentially of, or consist of any of the compound components in amounts/concentrations as disclosed, cited or inferred herein, including but not limited to any of the compound components in tables 1-7 and 10, and any structural isomers thereof, any such alcohol component, aldehyde component, ketone component, acid component and/or carbon and/or methylene homologs of the ester component, whether acid-derived and/or alcohol-derived portions thereof. Each such compound component or portion/substituent thereof, regardless of the amount/concentration thereof, has compositional and characteristic differences and can be used in the compositions and methods of the present invention in an amount/concentration that is independent of another such component or another compound component (or portion/substituent) or amount/concentration. Thus, it is to be understood that the compositions and/or methods of the present invention can be implemented or utilized in varying amounts or concentrations in the absence of any one of the component compounds (or portions and/or substituents thereof), which may or may not be specifically disclosed, referred to, or inferred herein, or which varying or absent may or may not be specifically disclosed, referred to, or inferred herein, as disclosed herein.
In a preferred embodiment, such biologically effective compositions of FFCs (prepared as liquid mixtures) are readily volatilized at room temperature and spread sufficiently in an enclosed space to effectively inhibit and/or kill undesirable contaminating fungi (mold) on surfaces that need to be free of such harmful microorganisms. This mixture can be applied as a spray (e.g., the ingredients under pressure) or simply placed in a container and allowed to evaporate in a closed container or sealed bag.
In any event, the FFC compositions of the present invention can be incorporated into a variety of end use compositions, limited only by their application. Such compositions include, but are not limited to, those relating to human/animal food or nutrition, personal hygiene, health care, agricultural, industrial, residential, medical, and oral applications. In certain non-limiting embodiments, the FFC composition and/or components thereof present may be from about 0.1 wt.% or less to about 99.9 wt.% or more of the particular end use composition. This level of addition is limited only by the desired antimicrobial effect and/or formulation factors.
The FFC compositions of the present invention are effective at effective dosage levels in killing many plant pathogens, fungi, which can cause food spoilage, microorganisms which can cause major human disease, and microorganisms which can contaminate work surfaces, homes, and other buildings. A non-exhaustive list of such applications is as follows:
1. for treating cheese in storage or preparation to control unsightly mold contamination of the surface and eventual spoilage of the cheese pieces.
2. For the treatment of various plant parts in storage, including roots, tubers, stems, seeds and other organs (organs) which can ultimately be used for food preparation, or for cultivation and re-planting or agricultural purposes.
3. For decontaminating buildings having a mouldy surface or infected with a mould which may be problematic.
4. For the conservation of refuse transported over long distances offshore from one port to another, which is eventually fermented into energy-related products.
5. For decontaminating soils which may carry potentially phytopathogenic microorganisms.
6. For the treatment of patients suffering from tuberculosis and other mycobacterial infections.
7. For treatment to control nasal infections and clean the nasal passages.
8. For incorporation into specifically designed polymers that can be used in packaging and thereby preserving materials, including food, fiber, and other items that require longer-term safe storage.
More generally, the compositions of the present invention may be used for organisms selected from the group consisting of fungi, bacteria, microorganisms and a range of other microorganisms or pests to inhibit their growth or kill them. Such compositions in amounts effective to at least partially kill or inhibit the growth of the organism may be contacted with the organism using methods well known to those skilled in the art. Alternatively, it may be used for the treatment of human or animal waste, for example as a waste water component, or for solids management or treatment. Such compositions may also be used to purify human and animal waste, for example to reduce or remove bacterial and fungal contamination. In another aspect, such compositions can be used to treat or prevent mold in a building material or structure by contacting the structure, the building material, or the space between the building materials in an effective amount thereof or the vapor thereof. For purposes of illustration only, an effective amount of such compositions may be used alone or in combination with other fumigants or actives indoors or during fumigation of an entire building.
For use in agricultural applications, the present invention provides methods of treating or protecting fruits, seeds, plants, or soil surrounding plants from infection by organisms such as fungi or bacteria by contacting the microorganisms with an effective amount of one or more of such compositions described herein.
As described above, the present invention provides methods for preventing, treating, inhibiting, and killing bacterial, fungal, viral, and/or other microbial infections. Such methods may comprise applying to an article, animal/mammalian or plant substrate, having such infection or growth or capable of supporting such infection or growth, an effective amount of a composition of the present invention alone or added to a composition or formulation. Accordingly, the present invention provides one or more compositions for pharmaceutical, personal care (e.g., without limitation, cosmetic), industrial, and/or agricultural applications.
Microbial treatment may be accomplished by contacting bacteria, fungi, viruses and/or other microorganisms with an effective amount of a composition of the present invention. The contacting can occur in vitro or in vivo. By "contacting" is meant bringing together the composition of the present invention and such microorganisms in a manner sufficient to prevent, inhibit and/or eliminate infection and/or growth of the microorganisms. The amount of such compositions effective for such treatment can be determined empirically and making such determinations is within the ability of those skilled in the art. Inhibition includes reduction and elimination of microbial growth/activity.
The compositions of the present invention may be administered or contacted with a human, animal or article substrate surface by any suitable route, including, but not limited to, orally or nasally (e.g., for pharmaceutical or personal care applications) and topically, such as by powder, granule, liquid, spray, salve, lotion or cream. Thus, the compositions of the present invention may comprise component compounds each in admixture with one or more acceptable carriers and optionally with one or more other compounds or other materials. Such carriers should be "acceptable" in the sense of being compatible with the other components/ingredients of the formulation and not deleterious to the desired effect or application.
Regardless of the route of delivery, treatment, or administration selected, the compositions of the present invention can be formulated by conventional methods known to those skilled in the art to provide acceptable concentrations or dosage forms. The amount or concentration of any such composition or component thereof, with or without a carrier, will vary depending on the target microorganism/substrate/article being treated, the particular mode of application/delivery, and all of the other factors mentioned above. The amount associated with the carrier material can generally be that amount of such composition which provides the lowest or minimum concentration effective to produce the desired antimicrobial effect.
As demonstrated in the examples below, the relative amounts or concentrations of the FFC composition and the other optional components of the composition in the compositions of the present invention can vary widely within effective ranges. The concentration and/or dosage used is preferably selected to achieve enhanced or improved activity over the individual prior art components alone and/or to maximize the activity of the composition at the lowest effective component concentration. Thus, the weight ratio and/or percentage concentration that results in this enhanced activity depends not only on the particular FCC composition used, but also on the particular end use of the composition, including but not limited to the climate, soil composition, nature of the substrate, article and/or microbial host to be treated and/or potential contact with a particular microorganism.
The method for preparing the formulation or composition comprises the step of bringing into association the composition or one or more component compounds of the present invention with the carrier and optionally one or more accessory ingredients. Typically, such formulations are prepared by bringing into association such compositions/components with a carrier (e.g., a liquid or finely divided solid carrier), and shaping the product as desired.
Whether the compositions of the present invention, or any article of manufacture into which such compositions are incorporated, the formulations related to the present invention may be in the form of capsules, cachets, pills, tablets, powders, granules, ointments (pastates), or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs (elixir) or syrups, or as lozenges (using inert substrates such as gelatin and glycerin, or sucrose and acacia), and the like, each containing a predetermined amount of a composition of the present invention or a component thereof.
In other solid such formulations (e.g., capsules, tablets, pills, dragees, powders and granules, etc.), the compositions of the present invention may be mixed with one or more other active ingredients and/or acceptable carriers, such as sodium citrate or calcium hydrogen phosphate, and/or any of the following: (1) fillers or extenders (extenders), such as starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarding agents (solution retaring agents), such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) a colorant. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (for example, gelatin or hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrating agents (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agents. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
Tablets and other solid forms of such compositions or articles into which such compositions are incorporated, such as dragees, capsules, pills and granules, can optionally be scored or prepared with coatings or shells, such as enteric coatings and other coatings well known in the formulation art. They may be formulated, for example, using hydroxypropylmethylcellulose at various concentrations to provide a desired release profile, other polymer matrices, liposomes and/or microspheres to provide sustained or controlled release of the active ingredient therein. These compositions may also optionally contain opacifying agents and may be of a composition that it releases the active ingredient(s) only, or preferentially, in a particular portion of the gastrointestinal tract, and optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form.
Liquid forms for use or administration of the present invention include pharmaceutically or otherwise acceptable emulsions, mixtures, microemulsions, solutions (including those in distilled or purified water), suspensions, sprays, syrups and elixirs. In addition to the compositions of the present invention or the compound components thereof, the liquid forms may contain inert or other diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Such compositions and/or related articles may also contain, in addition to the inert diluent, adjuvants such as, but not limited to, wetting agents, emulsifying and suspending agents (e.g., binders and spreaders for agricultural applications), colorants, perfuming agents, and one or more other preservatives. Suspensions may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide (aluminum metahydroxide), bentonite, agar-agar and tragacanth, and mixtures thereof.
In the present invention, formulations of the compositions of the invention for matrix or topical (e.g., in the personal care or hygiene product field) application/delivery and/or articles or products incorporating the compositions of the invention include powders, sprays, ointments, creams, lotions, gels, solutions, patches and inhalants. Such ointments, salves, creams and gels may contain, in addition to a composition of the present invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth and other gums, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Likewise, powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. The spray may additionally comprise a conventional propellant, for example a volatile unsubstituted hydrocarbon such as butane and propane, or be delivered under positive gas pressure.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and organic esters, such as ethyl oleate. For example, proper fluidity can be maintained, for example, by the use of coating materials (e.g., lecithin), by the maintenance of the required particle size in the case of dispersants, and by the use of surfactants.
Depot form articles or products incorporating the compositions of the invention may be prepared by forming a microencapsulated matrix of the active ingredient in a biodegradable polymer such as polylactide-polyglycolide. The release rate of the active ingredient can be controlled depending on the ratio of active ingredient to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations can also be prepared by encapsulating the active ingredient in liposomes or microemulsions which are compatible with body tissues.
In addition, the compositions of the present invention and/or articles or products incorporating such compositions may also contain other chemical and/or biological, multi-site and/or unit-site fungicidal or antifungal, antibacterial and antimicrobial agents having similar and/or different modes of action, as is well known to those skilled in the art. Such agents may include, but are not limited to, potassium bicarbonate, silica, copper or sulfur-based compounds, and/or vegetable oils (e.g., neem oil). Further, such agents may include, but are not limited to azoles; polyenes, such as amphotericin B and nystatin; purine or pyrimidine nucleotide inhibitors, such as flucytosine; polyoxins, such as heliomycin; other chitin inhibitors, elongation factor inhibitors, such as coprocostin (sordarin) and its analogs; mitochondrial respiration inhibitors, sterol biosynthesis inhibitors, and/or any other fungicidal or biocidal composition known to those skilled in the art to be suitable for treating or preventing yeast or fungal, bacterial, viral, and/or other microbial infections of plants, other substrates, animals, and/or humans, or found on or within any article of manufacture.
In certain embodiments, an article of manufacture or product incorporating the compositions of the present invention may further comprise one or more preservatives known in the art, including, but not limited to, sorbic acid or benzoic acid; sodium, potassium, calcium and ammonium salts of benzoic, sorbic, hydroxymethylglycolic and propionic acids; methyl, ethyl, propyl, and butyl benzoate (paraben) and combinations thereof.
The compositions of the present invention may contain compounds that contain acidic or basic functional groups and are thus capable of forming pharmaceutically or otherwise acceptable salts with pharmaceutically or otherwise acceptable acids and bases. The term "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid and base addition salts of such compounds. Regardless, such salts can be prepared by reacting such compounds with a suitable acid or base. Suitable bases include, for example, the acceptable hydroxides, carbonates or bicarbonates of metal cations, ammonia (ammonia) or, for example, the acceptable organic primary, secondary or tertiary amines. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. Representative acid addition salts include hydrobromide, hydrochloride, sulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthalate (napthalate), methanesulfonate, glucoheptonate (glucoheptonate), lactobionate, laurylsulfonate, and the like.
The compositions of the present invention can be used as aqueous dispersions or emulsions, since they can be diluted (e.g., with water or other fluid components) prior to use, and can be obtained in the form of concentrates containing high proportions of FFC (with or without surfactant) compositions. Emulsifiable concentrates or emulsions can be prepared by dissolving the compositions of the present invention, together with any other desired active ingredient, in a solvent optionally containing a wetting or emulsifying agent, and subsequently adding the mixture to water, which may also contain a wetting or emulsifying agent. Suitable organic solvents include alcohols and glycol ethers. These concentrates should preferably be able to withstand long term storage and, after such storage, be able to be diluted with water to form an aqueous formulation which remains homogeneous for a sufficient time to enable its application by conventional spray devices.
As is well known to those skilled in the art, depending on the type of end use application, the article or product into which the composition of the present invention is incorporated may also contain any other desired components, including, but not limited to, solid or liquid carriers to facilitate use; a surfactant comprising a biosurfactant; protective colloids, adhesives, thickeners, thixotropic agents, penetrants, stabilizers, chelating agents, texture modifiers, flavorings (e.g., for pre-harvest or processed food/beverage applications), sugars, colorants, and the like.
For example, such compositions and/or related articles or products may be used for agricultural purposes and formulated using such carriers or diluents. The composition, formulated or unformulated, may be applied directly to the foliage of the plant, to the seeds or other medium in which the plant is growing or is to be planted, or it may be sprayed, dusted or applied as an emulsion or ointment formulation, or it may be applied as a vapour or as slow-release granules. It may be applied to any part or vicinity of the plant, including the leaves, stems, branches or roots, or to the soil surrounding the roots, fruits or vegetables (pre-harvest or post-harvest), or to the seeds before planting, or substantially to the soil, or to irrigation water, or to hydroponic culture systems. The compositions of the present invention may also be injected into plants or sprayed onto vegetation (including fruits and vegetables) using low volume or pressure or electrokinetic spraying techniques or any other treatment methods known in the art or industry.
In certain embodiments, whether related to agriculture or food processing, the compositions and/or articles or products incorporating the compositions of the present invention may be in the form of dustable (dustable) powders or granules containing solid diluents or carriers such as fillers (also such as animal litter or cat litter, animal or cat litters), kaolin, bentonite, Diatomaceous earth (kieselguhr), dolomite, calcium carbonate, talc, powdered magnesia, fuller's earth, gypsum, Diatomaceous earth (Diatomaceous earth) and china clay. Such particles may be pre-formed particles (pre-formed granules) suitable for application without further treatment. These granules can be prepared by impregnating the filler particles with the composition of the invention or other active ingredient, or by granulating a mixture of active ingredient and powdered filler. For example, a composition for seed dressing (seed dressing) may comprise an agent (e.g., mineral oil) that aids in the adhesion of the composition to the seed; alternatively, the active ingredient may be formulated for seed dressing using organic solvents. The composition may also be in the form of a wettable powder or water dispersible granules containing a wetting or dispersing agent to facilitate its dispersion in a liquid. The powders and granules may also contain fillers and suspending agents. Alternatively, the composition may be used in microencapsulated form. They may also be formulated as biodegradable polymeric formulations to obtain a controlled slow release of the active substance.
Regardless, such solid formulations comprising the compositions of the present invention can provide a variety of products or articles in different forms, shapes, or moldings (molding), including, but not limited to, cylinders, rods, chunks, capsules, tablets, pills, small particles (also e.g., pet food), strips, and ears, and the like. Alternatively, the granulated or powdered material may be compressed into tablets or used to fill a variety of capsules or shells. As noted above, such compositions of the present invention, whether formulated or not, can be used alone, applied to a substrate or incorporated into a product or article for a variety of different end uses, including but not limited to pharmaceutical, personal care, industrial, and agricultural compositions, and related methods of use.
Examples of the invention
The following non-limiting examples and data illustrate various aspects and features associated with the compositions and/or methods of the present invention, including the preparation and use of antimicrobial compositions comprising various component compounds as described herein. The compositions and methods of the present invention provide surprising, unexpected and contrary results and data compared to the prior art. While the utility of this invention has been described in terms of the use of various compositions and component compounds useful in the invention, it will be appreciated by those skilled in the art that similar results can be obtained using a variety of other compositions and component compounds commensurate with the scope of this invention.
Example 1a
Separation of fungi: in 3 months of 2007, small stems of a number of mini pineapples (Ananas anaassoides) were collected from plants grown in the area of bauhinia, borlivia. It was collected from the tropical dilute grassland area adjacent to the rainforest at 12 ° 40 '07 "S and 68 ° 41' 58" W and immediately transferred for analysis. A plurality of small pieces (2-5 inches) were cut from the stems and placed in 70% ethanol under a laminar flow hood for 30 seconds. The stems were individually held in a flame using sterile forceps to remove excess alcohol. A small piece of internal tissue (tree bark) was cut out and placed on Potato Dextrose Agar (PDA) which was actively growing with m.albus isolate 620 on it, placed on a plate (plate) with a central hole cut out on one side. This technique can be used to efficiently select other Muscodor isolates (Worapong et al, 2001a & b). The dishes/plates (Petri plates) were periodically checked for any fungal growth during the two-week incubation period. Once hyphae were observed, hyphal tips were cut from agar under sterile conditions and placed on fresh PDA. The isolate is examined in this manner. Multiple Plates (PDA) were used to determine whether the fungus produced volatile antibiotics. The method comprises the following steps: a 1 inch portion of the agar was removed from the middle of the plate and a small piece (a plug of) of isolate was spread (plated) on one side and allowed to grow for several days, followed by spreading the test organism on the other side of the gap.
Example 1b
Classification of fungi: the fungi are essentially related to mini-pineapples (a. ananasoides) and are incomplete fungi (deuteromycetes) belonging to the group of the sporogenous fungi (mycelia sterilia). When cultured in the absence of direct sunlight, whitish Fungal colonies (Fungal colloids) were detected on all media. When placed in direct sunlight, a slightly peach-colored fungal colony was detected on all media. No Spores (Spores) or other fruit bodies (fruiting bodies) were observed under any conditions. Hyphae (Hyphae) (0.6-2.7 μm) usually grow through branches, sometimes forming a complete helix (ca.40 μm) and carrying cauliflower-like entities (3.5-14 μm) attached to it. All tested media were observed under all conditions, and newly developed hyphae grew in a wavy pattern (undulating pattern). Mycelium (Mycelium) on PDA covered the plate for 3-4 weeks and produced fruit odor.
Positive model specimen (Holotype): endophytes (endophytes) on mini-pineapples. The collection was performed in the Heath River region of the region of bauhinia amazonia. Positive model specimens were from a single mini pineapple stem (collected from the Heath River county). Muscodor crispans were deposited as viable cultures at the University of canada University of montage collection of viable fungi (Montana State University mycological collection) under accession number 2347 (2/29/2008). Both the 18S rDNA and ITS sequences of M.crispans (B-23) have been filed in GenBank under the accession number EU 195297.
Complete generation (Telomorph): similarity of 18S rDNA gene sequence data between M.crispans and Xylariaceae (Xylariaceae) based on GenBank databaseA complete generation of this fungus can be found in the family Calycoridae (Bruns et al, 1991; Reynolds and Taylor 1993; Mitchell et al, 1995; Guarro et al, 1999; Taylor et al, 1999). Molecular data from the m.crispans 18S rDNA gene sequence showed 620100% homology to m.albus isolate.
Lexical source (Etymology): the generic name Muscodor is from Latin and has the meaning of moldy (musty). This is consistent with the odor profile produced by the first three isolates in this genus. The species crispan is from Latin and means "coiled, wavy". Hyphae grow in a regular wave-like pattern.
Example 2a
Scanning electron microscopy: following the procedure described in Castillo et al (2005), the isolate of example 1 was examined by scanning electron microscopy. Agar blocks and host plant blocks supporting fungal growth were placed on filter paper bags, then placed in 0.1M sodium cacodylate buffer (pH 7.2-7.4) containing 2% glutaraldehyde (containing the wetting agent Triton X100), aspirated (asparated) for 5 minutes and left to stand overnight. The next day, it was washed 6 times for 15 minutes each in water buffer 1: 1, then washed 15 minutes in 10% ethanol, 15 minutes in 30% ethanol, 15 minutes in 50% ethanol, 5 times for 15 minutes each in 70% ethanol, then left to stand overnight or longer in 70% ethanol. Subsequently, the washing was performed 6 times for 15 minutes in 95% ethanol, followed by 3 times for 15 minutes in 100% ethanol, followed by 3 times for 15 minutes in acetone. The microbial material was dried at the critical point, gold sputter coated (gold sputter coated) and the image recorded by XL30 ESEM FEG in high vacuum mode using an Everhart-Thornley detector. Hyphae were measured using Image J software available on-line.
Example 2b
Biology of fungi: the fungus produces white mycelium on water-based media. No fruiting body structure or spores of any type were found under any laboratory conditions. Hyphae tend to intertwine to form a helix. Other species of Muscodor also have this tendency (Worapong et al, 2001 a). Newly developed hyphae tend to grow in a wavy manner (not a typical vertical manner) and are often intertwined to form a rope-like structure. This growth pattern can be demonstrated to be a diagnostic tool for identifying this organism in vivo vaccination studies. This fungus also produces cauliflower-like structures that appear to be connected to the hyphae by small segments (strands). These entities did not germinate under any conditions and thus did not appear to be spores. This observation appears to be unique to Muscodor spp and is currently largely unnoticed in any other fungal species.
Example 3a
Growth and storage of fungi: it was determined that when several carnation leaves were placed on top of the actively growing isolate to promote sporulation, the isolate did not produce spores or any other fruit bodies, and no such structure was observed after one week of incubation at 23 ℃. The fungi were additionally plated on a variety of different media including Cellulose Agar (CA), Malt Agar (MA) and Corn Meal Agar (CMA) to determine if sporulation was indicated. No other distinct fungal characteristics were found except for the slower growth rate on some media, and no fruiting bodies or spores were observed.
Isolated fungi are stored in pure culture form using a variety of methods, one of which is the filter paper technique. The fungus was also grown on PDA, which was subsequently cut into small blocks, placed in vials containing 15% glycerol and stored at-70 ℃. The fungi were also stored at 4 ℃ by a similar method using distilled water instead of glycerol. However, the most effective method of storage is on sterile barley seeds grown at-70 ℃.
Example 3b
Other more typical characteristics of isolated m.crispans were also examined and compared to m.albus. The Muscodor crispans produced slow-growing, dense white mycelium on all media tested unless placed in direct sunlight (resulting in the mycelium exhibiting a pale pink color). In contrast, m.albus produced whitish mycelium in all similar media and test conditions (wortpog et al, 2001 a). Early hyphae also grew in a wavy fashion, rather than the typical linear-like fashion common to m.albus (Strobel et al, 2001). Spores were not formed on any of the media including the media containing the host plant material or carnation leaves. Hyphae vary in diameter (0.8-3.6 μm) and often intertwine to produce more complex structures, even hyphal helices (FIGS. 1-3). These hyphae are generally larger than those of m.albus (wortong et al, 2001 a).
Example 4
Qualitative analysis of volatiles: the method used to analyze the gas in the airspace above the 10 day old mycelium culture in the plates was similar to that used for the original isolate of strain m. First, a baked "solid phase microextraction" syringe (Supelco) consisting of 50/30 divinylbenzene/carburen placed on polydimethylsiloxane on a stable elastic fiber (flex fiber) was placed through a small hole drilled in the side of the growth plate that supports (transporting) the fungus. The fibers were exposed to the gas phase of the fungus for 45 minutes. Subsequently, the injector was inserted into the splitless injection port of a Hewlett Packard 6890 gas chromatograph containing a 30m x 0.25mm I.D.ZB Wax capillary column with a film thickness of 0.50 mm. The column temperature was programmed as follows: 30 ℃ for 2 minutes, then warmed to 220 ℃ at 5 ℃/minute. The carrier gas was ultra-high purity helium (regional distributor) with an initial column head pressure of 50 kPa. The fibers were conditioned for 20 minutes at 240 ℃ under a stream of helium gas before trapping volatiles. It is used for 30 secondsTo add sample fibers to the GC. The gas chromatograph was connected to a Hewlett Packard 5973 mass selective detector (mass spectrometer) operating at unit resolution. Data acquisition and data processing were performed on a Hewlett Packard ChemStation software system. Initial identification of compounds in fungal volatilization mixtures was performed by library comparison using NIST database.
Example 5a
Isolation and acquisition of ITS-5.8S rDNA sequence information from fungal DNA: using fast homogenization: plant leaf DNA amplification kit (Cartagen; Washington, USA) 10-day-old cultures of the fungus of the invention grown on PDA after incubation at 25 ℃ were used as the DNA source. Some of the techniques used are similar to those of the genetic characterization of other m.albus isolates from australia Ezra et al, 2004). Cut out a square (0.5 cm) of cultured mycelia from one week-old culture2). The agar was scraped from the bottom of the block to remove as much agar as possible. The pieces were placed into a 1.5ml Eppendorf tube and incubated at about-80 ℃ for about 10 minutes. Subsequently, the DNA was extracted according to the instructions of the kit manufacturer. The extracted DNA was diluted in double-distilled sterile water (1: 9) and PCR amplification was performed using 1. mu.l of the sample. The ITS1, 5.8S ITS2 rDNA sequences were amplified by polymerase chain reaction using primers ITS1(TCCGTAGGTGAACCTGCGGG) and ITS4 (TCCTCCGCTTATTGATATGC). In a PCR mixture containing 1. mu.l of DNA extracted from fungal cultures (1: 9 dilution), 0.5. mu.l of primer ITS1 and 0.5. mu.l of primer ITS4, 7. mu.l of RedMixTM, and 1.5mM MgCl2(GeneChoice, Inc., Maryland, USA) and 5. mu.l of PCR-grade ddH2The PCR procedure was performed in 14. mu.l reaction mixture of O (Fisher Scientific, Wembley, Western Australia, Australia). PCR amplification was performed in a Biometra personal cycler (Goettingen, Germany): 96 ℃ for 5 minutes, followed by 35 cycles of 95 ℃ for 45 seconds, 50 ℃ for 45 seconds, and 72 ℃ for 45 seconds, followed by 5 minutes at 72 ℃. TAE buffer (GelXLUultra V-2 from Labnet International, Inc. (Woodbrid, NJ, USA)) or Wealtec from Wealtec Inc. (Georgia, USA) was usedGES cell system, PCR products were detected using gel electrophoresis at 100V on a 1.3% agarose gel for 30 minutes. The gel was soaked in 0.5. mu.g ml-1 ethidium bromide solution for 5 minutes, followed by washing with distilled water for 5 minutes. Gel Imaging was performed under UV light in a bioimaging system (model 202D; DNR-Imaging Systems, Kiryat Anavim, Israel). A PCR product of 500bp was purified using UltraClean PCR Clean Up DNA purification kit (MO BIOLABORIERS, Inc., California, USA). The purified product was subjected to direct PCR sequencing. The ITS1 and ITS4 primers were used to sequence the double strands of the PCR product. Sequencing was performed on a MegaBACETM1000 analysis system (Danyel Biotech Ltd., Rehovot, Israel) using a DYEnamic ET terminator. The sequences were submitted to GenBank on the NCBI website. The sequences obtained in this study were compared to the GenBank database using BLAST software on the NCBI website.
Example 5b
Molecular biology of Muscodor crispans: partial sequences of 18S rDNA, ITS1, 5.8S and ITS2 have been shown to be highly conserved regions of DNA and are therefore very useful in biological taxonomy (Mitchell et al, 1995). The discriminating partial sequences on these molecules of m.crispans were obtained and compared to the data in GenBank. After searching for the 18S rDNA sequence, a high-level BLAST search was performed on the 525bp sequence of m.crispans. The results show 100% identity with 525bp (AF324337) of m.albus. Partial ITS1 to m&Comparative analysis of 2 and 5.8S rDNA sequences showed 95%, 90% and 91% homology with ITS1 and 2 of M.albus (AF324336), M.roseus (AY034664), Xylella enterobacter (X.enteroleuca) CBS 651.89(AF163033), X.arbuscula CBS 452.63(AF163029) and C.schizophyllum (Hypoxylon fragrans) (HFR246218), respectively.
Example 5c
Although part of the description of the invention relates to isolated novel fungi, variants and mutants of this fungus are also included within the present disclosure as understood in the art. The terms "variant" and "mutant" may be according to the definitions provided in U.S. patent No.6,911,338, which is incorporated herein by reference in its entirety. The present invention may therefore relate to variants or mutants of m.
Example 6a
Bioassay of crispans against plant pathogens. Crispans' volatile by-product vapors were tested for microbial inhibitory activity using a relatively simple test as described in the literature previously (Strobel, et al, 2001). Agar strips (2cm wide) in standard PDA plates were removed, inoculated with m. Subsequently, test fungi or bacteria were inoculated on the other side of the plate using a small agar plug for fungi. Bacteria and yeast were streaked (streamed) onto agar (1.5cm long). Subsequently, a piece of parafilm was used to cover the plate and incubated at 23 ℃ for 48 hours. First, the effect of m.crispans on the growth of the test organisms was identified by examining the presence or absence of growth at the inoculation site. If growth is observed, the diameter of the fungal hyphae at both locations is measured. The biological activity of the steam on fungi and yeast was assessed by assessing the extent of influence of the growth of fungi and yeast (growth percentage) on the control plates (Strobel et al, 2001). If no growth was observed, at some point after exposure to the vapor, the test organisms were removed from the test plates and inoculated onto fresh PDA plates under sterile conditions to determine the viability of the test organisms.
Using the above method, it was demonstrated that volatile by-products of fungi are lethal to a variety of fungi and bacteria when m.crispans are grown on PDA at 23 ℃ for 7-10 days. Gram-negative and gram-positive bacteria, as well as yeasts and each of the main types of fungi, are used as test organisms. Most of the test organisms were 100% inhibited and died after 2 days exposure to the m.crispans byproduct (see table 8). Some test organisms did not die after 2 days exposure to m.crispans volatiles, but their growth was significantly inhibited by volatile byproducts and died after 4 days exposure. Such organisms include: penicillium roquefortii (Penicillium roquefortii), Helminthosporium putamen (Bipolaris sorokiniana), Septoria septoria (Stagonospora sp.), Fusarium oxysporum (Fusarium oxysporum), and the like.
Table 8: effects of crispans volatile by-products on various plant fungal pathogens and some miscellaneous bacteria (aborobacteria). This inhibition value was calculated as% growth inhibition compared to untreated control test organisms. The test was repeated at least 3 times with similar results. Inhibition of test organisms was recorded 48 hours after exposure to fungi and volatile fungal by-product vapors.
*Note that: these organisms were drawn into test plates and growth was indicated if colony development eventually occurred. Crispans' volatile by-products after appropriate exposure, streaked areas were compared to growth on control plates and% inhibition assessed. Finally, each organism was re-scored on PDA plates to test for viability.
Example 6b
Referring to table 8, the effect of volatile by-product vapors of m.crispans on Botrytis, especially Botrytis cinerea (b.cinerea) which causes gray mold of various plants, is particularly significant. The inhibiting and killing action is also applicable to Botrytis allii, which causes Botrytis allii, Botrytis, Botr. This result indicates that the present invention can effectively change the surface or storage environment of the harvested product to prevent mold and related events, but is not limited thereto. Again, this result supports the use of the FFC compositions of the invention in treating onion (e.g., Vidalia onion, Vidalia onions), shallot and garlic products to prevent or control fungal growth.
Example 6c
The vapors from m.crispans volatiles are also effective against many spoilage-causing fungi and fungi that grow on grains (e.g., corn, wheat, barley, rice, etc.), and the present invention can also be used with a variety of fruits and vegetables, such as potatoes, beets, carrots, sweet potatoes, such as grains, fruits or vegetables, before and after harvest, in storage or in transit. Thus, the compositions and methods of the present invention are applicable in some areas in the agricultural and food processing fields related to most fungi and can be used for target organisms such as, but not limited to, Alternaria (Alternaria), Cladosporium (Cladosporium), Aspergillus (Aspergillus), Penicillium (Penicillium), Diplodia (Diplodia), Fusarium (Fusarium) and Gibberella (Gibberella). (see, e.g., Table 8.)
Example 6d
The vapor from the m.crispans byproduct was effective against mycosphaerella fijiensis (mycosphaeraseliajis) fungus (see table 8). Thus, the present invention is useful for treating banana and plantain fungus-related leaf spot (Black Sigatoka disease).
Example 6e
Citrus canker disease (Citrus canker disease) threatens the death of the Citrus industry in the united states. As shown in table 8, the vapors from the m.crispans byproduct were effective in killing the canker causing pathogenic Xanthomonas carpet citrus var. This result indicates that the FFC compositions and related methods of the present invention can be effectively used to treat seeds, seedlings, orchards, equipment or apparatus (including, for example, worker's equipment and clothing) and/or harvested fruit to prevent, inhibit or control canker.
Example 7
Following the tests and results of example 6, bioassays of vapors of volatile by-products of m.crispans against a variety of other plant and human pathogenic fungi and bacteria were performed (see table 9 below). The fungi were grown on quarter-circle X-plates with PDA and incubated at room temperature for 3-5 days before inoculation with one or more test organisms. Control plates were prepared at the same time as inoculation and grown on the same media that was optimal for the test organism individuals. The test organisms, Staphylococcus aureus (Staphylococcus aureus 6538), Salmonella choleraesuis (Salmonella cholerasuis 10708), Escherichia coli (Escherichia coli 11229), Staphylococcus aureus ATCC 43300(MRSA), and Vibrio cholerae (Vibrio cholerae ATCC 14035) were grown on Tryptone Soy Agar (TSA) in the remaining three quarters of the X-plate. Three plates of each organism and appropriate controls were exposed to vapors of fungal by-products for about 2, 4 and 6 days at room temperature. To test the viability of the test microorganisms, the fungi were then physically removed and the control and test plates were placed in an incubator at 35 ± 1 ℃ for at least 3-4 days, but the mycobacteria (Mycobacterium spp.) were incubated for an additional about one month. This is to determine whether the vapors of the byproducts have inhibited or killed the test organisms and to assess the viability of the organisms. The same procedure was performed for Yersinia pestis (Yersinia pestis) and Bacillus anthracis (Bacillus anthracaris) but with the contact time changed to 3 days and 5 days, and Yersinia pestis was exposed to the fungus at 28 + -1 deg.C with 5% CO2And (3) incubating. Using the procedure described above, M.marinum (Mycobacterium marinum ATCC 927) was grown in the remaining three quarters of a circle of 7H11 agar (Difco Co) and cultured at 33. + -. 1 ℃. All three replicates of each organism performed identically in the test.
For all M.tuberculosis (Mycobacterium tuberculosis) strains that were also grown on 7H11, a portion of the agar was removed from the plate and inserted into the B-23 fungus (on PDA). Subsequently, the broth culture was inoculated to the plate. Control plates without the presence of fungi were also inoculated. At each designated time interval, portions of agar were removed from the plates, transferred to individual empty plates, and placed in an incubator at 35 ± 1 ℃ to determine the viability of the microorganisms. The plate was placed in a plastic bag with a wet wipe to prevent drying.
Pseudomonas aeruginosa (Pseudomonas aeruginosa 15442) and Burkholderia thailandiensis 70038 were both grown on TSA agar. It was kept at room temperature to obtain the optimal growth temperature for the organism, and subsequently transferred to an incubator at 35 ± 1 ℃ and observed. Note that all tests using human pathogens were performed under strict and federally government approved biosafety conditions. All tests on human pathogens were repeated at least twice.
Table 9: effect of crispans volatile by-products on various gram-positive and gram-negative bacterial species. The contact time varies with the particular target organism, and the viability of the test organism (labeled as growth or no growth) is determined after that period.
As shown in table 9, all four acid fast bacteria (mycobacterium tuberculosis strains) were killed 2, 4,7 and 14 days after exposure to actively growing m.crispans (6-10 day old cultures). Other bacteria that are killed after at least 2 days of exposure to m.crispans are: staphylococcus aureus 6538, mycobacterium marinum, yersinia pestis, and salmonella choleraesuis. Crispans exposure is relatively less affected or not affected at all the following: pseudomonas aeruginosa, Burkholderia tympani, Staphylococcus aureus (MRSA), Escherichia coli, Vibrio cholerae and Bacillus anthracis. However, the growth of staphylococcus aureus (MRSA) is only small mucous membrane and not any obvious colony, and thus, it is affected by VOC of m. In addition, the bacillus anthracis plates only left a few colonies on the contact plates, but after removal of m. Therefore, it is suspected that vapors of the m.crispans byproduct are only effective on vegetative cells (vegetitive cells) of bacillus anthracis, but do not act on spores. One month after the last observation period (14 days), no growth was observed on any of the plates exposed to the fungus, but growth was observed on all of the control plates.
The following tests of the examples illustrate various embodiments of the compositions of the present invention and their utility. A representative composition is provided in table 10, but without limitation as to the amount, concentration or ratio of ingredients. In certain embodiments, the amount of isobutyric acid can be replaced with an equal or approximate level of propionic acid. In certain such or other embodiments, ethanol may be replaced by acetic acid and/or 2-butanone may be replaced by acetic acid or propionic acid. In addition, various esters can also be substituted with isomers or homologs of the listed esters (e.g., but not limited to, 3-methylbutyl ester of propionic acid with 2-methylbutyl ester thereof). The results observed in the following examples were obtained from the compositions of the compounds listed in table 10. Consistent with this, a variety of other compositions may be used to achieve similar effects.
Table 10: food and flavor compound compositions useful for controlling harmful microorganisms
| Compounds from the series of FFCs* |
| Acetaldehyde |
| Ethyl acetate |
| 2-butanone |
| Propionic acid, 2-methyl, methyl ester |
| Ethanol |
| Acetic acid, 2-methylpropyl ester |
| Propionic acid, 2-methyl-, 2-methylpropyl ester |
| 1-propanol, 2-methyl- |
| 1-Butanol, 3-methyl-, acetate |
| Propionic acid, 2-methyl-, 2-methylbutyl ester |
| 1-butanol, 3-methyl- |
| Propionic acid, 2 methyl |
| Acetic acid, 2-Phenylethyl ester |
*Each of these compounds is liquid at room temperature and one of them can be used with the other to provide a liquid composition that is easily volatilized at room temperature or at a temperature and pressure that allows volatilization.
FFC compositions for controlling plant diseases
Example 8a
The relative ability of FFC to inhibit and kill test organisms was measured. Test solutions were prepared by placing the compounds into vials in the relative proportions given in table 10. The test mixture (20 microliters) was placed in a pre-sterilized cuvette (4x6mm) centered on the plate containing the PDA. When not used, the mixture was stored at 0 ℃. Newly grown and cut to 3mm3Test organisms (as described in table 9) on agar blocks (at least 3 agar blocks per test fungus) were placed 2-3cm from a small cup and the plate was wrapped with two layers of parafilm. After a given period of time, the growth of mycelium from the edge of the agar block was measured. However, in the case of Geotrichum candidum (Geotrichum candidum), it was streaked and examined for new visible growth and viability by re-streaking from the original area of the inoculated agar plate. An appropriate control was also set up in which no test solution was placed in the cuvette. At least two tests were performed on 20 μ l FFC mixtures and similar results were obtained.
Example 8b
The viability of the test microorganisms was checked by removing small agar blocks under sterile conditions, placing them on PDA plates and observing growth after 1-3 days, or by re-streaking Geotrichum candidum onto fresh PDA plates. The viability of the microorganisms can be assessed in this manner. The results shown in table 11a indicate that the organisms listed below are all inhibited by a particular FFC composition and are killed in most cases by contact with the FFC composition. These organisms include: aspergillus niger (Aspergillus niger), Penicillium sp. on cheese, Ceriporiopsis exigua (Ceriporia betacella), Verticillium dahaliae (Verticillum dahaliae), Pythium ultimum (Pythium ultimum), Phytophthora palmae (Phytophthora palmivora palmi), Mycophaeria fijiensis (Mycophaeria fijiensis), Rhizoctonia solani (T Rhizoctonia solani), Aspergillus fumigatus (Aspergillus fumigatus), Geotrichum candidum, Trichoderma viride (Trichoderma), Ganoderma lucidum (Ganodermata sp.), Curvularia (Curvularia sp.) and Boytisaria solani (Boytisallii). Thus, when properly applied, FFC compositions have the ability to control these pathogenic microorganisms. This result indicates that many other pathogenic microorganisms can be inhibited or killed by this mixture.
Table 11 a: a brief catalog of various phytopathogenic microorganisms and their sensitivity to representative FFC compositions of the present invention, wherein 20 microliters of the mixture is contacted for 2 days at 23 ℃ on Potato Dextrose Agar (PDA) in parafilm-sealed plates. Finally, the agar plugs with the test microorganisms were tested for viability after removal and placement on normal culture plates of PDA.
| Testing organisms | Influence on growth | Survival or death after 48 hours |
| Aspergillus niger | No growth | Death was caused by death |
| Penicillium on cheese | 95% inhibition | Survival |
| Cercospora farinosa | No growth | Death was caused by death |
| Verticillium dahliae | No growth | Death was caused by death |
| Pythium ultimum | No growth | Death was caused by death |
| Phytophthora palmae | No growth | Death was caused by death |
| Mycosphaerella fijiensis | No growth | Death was caused by death |
| Rhizoctonia solani | No growth | Death was caused by death |
| Aspergillus fumigatus | No growth | Death was caused by death |
| Geotrichum candidum | Without inhibition | Survival |
| Trichoderma viride | 60% inhibition | Survival |
| Ganoderma lucidum | No growth | Death was caused by death |
| Curvularia lunata (berk.) Kuntze | No growth | Survival |
| Testing organisms | Influence on growth | Survival or death after 48 hours |
| Green onion rotten grape spore fungus | No growth | Death was caused by death |
Example 8c
Referring to the data of table 11a, the activity spectrum of the FFC composition used demonstrates a different and/or enhanced antimicrobial effect compared to the vapors of m.
Example 8d
With reference to the above examples, and using similar techniques and procedures, the same pathogens were treated with propionic acid vapor. The comparative results are shown in table 11B below, where the data of table 11a are reproduced in columns a and B, and the observed effect of propionic acid alone is provided in column C. At 20 μ l, the amount of propionic acid was comparable to the propionic acid level in the present embodiment. Propionic acid is representative of a number of individual compounds known in the art to have certain antimicrobial effects. However, as shown by the comparative data in table 11b, the novel synergistic results provided by the compositions of the present invention are higher than and beyond those expected to be produced independently by the individual prior art components outside the context of the present invention. As shown in the table, the prior art is only inhibitory at best, whereas the compositions of the present invention eliminate (i.e., kill) many of the test pathogens. Similar results were obtained by comparison with other such individual prior art compounds/compositions.
Table 11 b: comparative results showing antimicrobial activity higher than propionic acid
*100% inhibition, but viability (ND) was not determined.
Use of FFC compositions for treating tuberculosis and other human pathogens
Example 9a
Isolates of mycobacterium tuberculosis (5901867, 50001106, 59501228, and 3081) of four clinically resistant strains were contacted with the FFC compositions. For each isolate, 10 μ L of the culture was placed in the middle of a 7H11 agar plate and spread evenly over the entire surface of the plate using a sterile plastic ring. The lid (small lid) of a 0.65ml microcentrifuge tube was cut and autoclaved for 15 minutes at 121 ℃ in an autoclave tube with a screw cap. The small lid was removed using sterile forceps and placed in the center of the inoculation plate. Control plates (one for each isolate) were not fitted with small lids. Three plates were prepared for each isolate and 5, 10 or 20 μ L FFC was placed in each of the three lids of each plate. Subsequently, the plate was placed in a zip-lock plastic bag with wet paper towel and incubated at 36 ℃. + -. 1 ℃ for about 28 days. After about 48 hours of contact, the flap is removed and discarded, and the plate is returned to the incubator. The paper towels were inspected from time to time and rewetted to prevent media dehydration. All control plates had growth. All plates exposed to 5. mu.L and 10. mu.L volatiles had growth. Only one isolate (50001106) exposed to 20 μ L of volatiles grew. It is noted that each Mycobacterium tuberculosis isolate is a clinically resistant strain of the organism. All tests were conducted under biosafety laboratory conditions approved by the U.S. government.
Control plates and plates exposed to 5. mu.L and 10. mu.L volatiles were plated at 4 months and 14 days 08 years. Plates exposed to 20 μ L of volatiles were plated at 4 months and 22 days 08. All plates were examined multiple times. The last examination was performed at 5/19 days 08, and those organisms that did not survive were labeled "- -", in table 12.
Table 12: inhibition effect of FFC on growth of drug-resistant mycobacterium tuberculosis
| Isolates of Mycobacterium tuberculosis | 5μL | 10μL | 20μL |
| 5901867 | + | + | -- |
| 50001106 | + | + | + |
| 59501228 | + | + | -- |
| 3081 | + | + | -- |
The actual effect of the FFC composition of the invention on another TB strain is shown in figure 1: the killing effect of FFC on drug-resistant Mycobacterium tuberculosis strain (110107). The left plate is a control plate that has not been treated with 20 microliters of FFC for 48 hours, and the right plate is treated for 48 hours, after which both plates are incubated at 36 ℃ for 28 days. From these experiments it is clear that FFC is able to kill 3/4 mycobacterium tuberculosis drug-resistant isolates. Currently, there is the prospect of using this FFC composition to treat tuberculosis in animals and ultimately conducting human trials.
Example 9b
In keeping with the data of the previous examples, the broad aspect of the invention can be demonstrated. Viable cultures and suitable media are prepared using materials and techniques well known to those skilled in the art. For example, contact with an FFC composition of the invention (e.g., by direct contact with a liquid composition or vapor thereof) can result in growth inhibition or death of the following e.coli-like bacteria (gram strains and morphologies): escherichia coli (gram positive, coryneform), Salmonella enteritidis (gram negative, coryneform), pseudomonas aeruginosa (gram negative, coryneform), staphylococcus aureus (gram positive, cocci), and listeria monocytogenes (gram positive, coryneform).
Likewise, such results have also been demonstrated to be obtainable using a variety of other gram-negative and/or gram-positive bacteria, such as, but not limited to, bacillus cereus (gram-positive, rod-shaped) and clostridium botulinum (gram-positive, rod-shaped).
Example 10
Crispan IC was calculated for artificial compositions that mimic volatile byproducts of m50(see Table 1). Referring to table 12, all test organisms were 100% inhibited using 15 μ L of the artificial mixture, and several of them were killed using as little as 10 μ L. Even with the largest volume of the mix (30. mu.L), Verticillium dahliae (Verticillium dahliae), Botryis cinerea (Botryis cinerea) and Aspergillus fumigatus (Aspergillus fumigatus) were not killed, but all three were 100% inhibited by either 10. mu.L or 15. mu.L of the test mix. The most sensitive organism is Pythium ultimum, which is killed at 10. mu.L and inhibited by 100% at 2.5. mu.L, in fact, since Pythium ultimum and Botrytis cinerea have essentially the same IC50But one is killed and the other is not (Table 13), so that the IC is50The values do not necessarily reflect the killing activity of the volatiles.
Table 13: IC of artificial mixtures of volatile by-product components of crispans for a variety of plant pathogens50. Sterile plastic tubes in the center of the test plate were added with a mixture ranging from 1 to 30 μ L and pathogenic organisms were placed near the edges of the plate. Viability was assessed after 48 hours and compared to control plates without the addition of the mixture but with sterile tubes in place. Any organisms that did not show growth after the period were identified as 100% inhibition, and organisms that did not show growth after 48 hours and did not grow immediately after isolation on PDA after 48 hours evaluation were identified as deadAnd (7) death. IC was determined by dividing the amount of artificial mixture required to cause 50% inhibition (μ L) by the total air gap in the plate (50mL)50The calculated value of (a).
Use of FFC compositions for treating waste to control microbial spoilage
Example 11
An artificial mix of items often regarded as litter is loaded into two cartridges (ammo cartridge boxes). These items consist of waste grains, flower parts, meat waste, newsprint fibers and various other waste. A small beaker containing 0.2ml of the above FFC composition was placed in one box. Put a beaker without FFC in another box. Both cassettes were incubated at 80 ° f for 10 days. At the end of said time, the box is opened and tested. No spoilage was evident in the cassette with FFC. On the other hand, the control box had been completely converted to a large amount of putrefaction. The use of FFC compositions in the treatment of waste is to keep the waste intact from spoilage when transported, to facilitate worldwide opportunities for fermentation of the waste into energy related products (e.g., methane). Figure 4 shows that the FFC composition protects the waste from microbial spoilage under this test condition.
Use of FFC compositions for treating cheese to control fungal spoilage
Example 12
Placing and/or soaking a vial containing the FFC composition described above in and/or with a clear plastic SaranThe block of packaging material is 10x 10 inches. Dipping plastic packaging material in FFC compositionSoak for 6 days, drain, then as the full inoculation penicillium sp cheese strain cheese sheet outside the packaging material. In another test, the cheese pieces were inoculated with fungi, followed by normal SaranThe packaging material was wound and then 10 microliters of FFC were injected. Suitable controls were prepared as described above with penicillium only, treated packaging material only, FFC alone and control (no treatment). The test cheese pieces were incubated at room temperature for 1 week, and then taste testing was performed on each cheese portion by laboratory personnel. Note that storage in this manner had no adverse effect on the taste of the cheese when compared to fresh slices of cheese that had been stored in the refrigerator. The cheese pieces that were completely infested with fungus were not eaten. As is evident from fig. 2, the use of the FFC composition under the packaging material or the use of the treated packaging material actually completely protected the cheese pieces from cheese spoilage and colonization by penicillium. The same occurs in the treated wrapper and the injection of 10 microliters of FFC under plain Saran wrapped cheese alone.
Use of FFC compositions for treating food and plant parts (e.g. plant products) to control fungal spoilage
Example 13a
Multiple yams were obtained for these experiments. The amount of surface contaminating microorganisms that ultimately cause spoilage is believed to be sufficient for inoculation. Thus, two pieces of yam were placed in a plastic box with a lid and sealed in the presence of a small beaker containing 0.2ml of FFC. The control box contained a beaker without FFC. Subsequently, the sealed cartridge was kept at room temperature for 10 days, followed by detection. As shown in fig. 3, it is evident that the treated yam pieces developed no surface and deeper stains, while the control yam developed surface stains and early decay (insipientdecay) in various areas: untreated yams are on the left side and those treated by FFC are on the right side. Note that a large area of fungal spoilage on the top end of the yam on the left side.
Example 13b
As a related end use application, the FFC composition and/or components thereof may be applied to the harvested fruit or vegetable product to compensate for the removal of any natural, waxy or protective coatings thereon. For example, harvested squash and similar products can be treated with FFC compositions (e.g., spray coating) to control/inhibit microbial growth, improve marketability, and extend shelf life.
Example 14
The synthetic FFC compositions of the invention (according to those described in tables 2-7 and 10) were successfully compared with the use of viable m.albus to control beet (Beta vulgaris L.) seedling stage diseases caused by pythium ultimum, Rhizoctonia solani AG 2-2 (Rhizoctonia solani) and pythium aphanidermatum (Aphanomyces cochlioides) and root knot nematode disease (meloidogyneic) on tomato (Lycopersicon esculentum). The synthetic composition provided equivalent control of damping off (damping off) to a starch-based formulation of live fungi for all three beet pathogens and significantly reduced the number of root gall on tomato roots. Rate studies using FFC compositions showed concentrations of 2. mu.l/cm3And 0.75. mu.l/cm3The soil carrier/culture medium components of (A) provide good control over rhizoctonia solani and pythium rhizoctonia rot of sugar beet, respectively. The concentration was 5. mu.l/cm3The sand provided 100% mortality to meloidogyne incognita within 24 hours. By comparison, using in vitro studies, this same rate of bio-rational agent (biological) provides a ratio of 5. mu.l/cm to the application3M.albus infested ground barley preparation of sand had less root knot gall.
Example 15
Corynebacterium melleus (Corynebacterium microorganisum) causes severe tomato loss through tissue withering and decay. An authentic culture of this bacterium was streaked into nutrient broth agar and a small cup was placed in the center of the plate. In the cup was placed 20 microliters of the manually laboratory prepared FFC composition of the present invention. Control plates contained no FFC composition. The plates were incubated for 24 hours and then assayed. There was no bacterial growth on the FFC-treated plates (see figure 5). As such, the FFC compositions of the present invention may be used to treat tomato seeds, plants or products, but are not limited thereto. Alternatively, the FFC composition may be mixed with water as pre-bed soil wetting (pre-bed soil drench).
Example 16
With reference to the foregoing and in accordance with the various embodiments herein before, the FFC compositions of the present invention may be used for prophylactic use or for the treatment of active disease states including, but not limited to, diseases affecting sugar beets, tomatoes, onions, cereals, bananas and plantains, as well as citrus crops and the like.
More broadly, the compositions and methods of the present invention can be used to treat and improve the viability of seeds, plants, products and/or related food products, whether disease prevention or in the presence of fungal or bacterial microorganisms, and whether the stage of the life cycle of infection (e.g., zoospores, etc.), development, growth or extent. Thus, one skilled in the art will appreciate that this composition can be contained and/or applied to the surface of or in contact with a seed, seedling, or plant (e.g., root, stem, leaf, etc.) or product thereof (e.g., pre-harvest or post-harvest) in any form (e.g., powder, granule, liquid, spray, suspension, vapor, ointment, gel, coating, etc.).
Example 17
The FFC compositions and/or components thereof, alone or in addition to various other compositions, can be used in a variety of end uses in the poultry, product and related food processing industries. The following examples provide a variety of such non-limiting applications.
Example 17a
A range of egg products, including but not limited to whole eggs and liquid whole eggs, additive whole eggs (formed whole eggs) and additive liquid whole eggs, salt whole eggs and liquid salt whole eggs, sugar whole eggs and liquid sugar whole eggs, and mixtures of such products (whether liquid or not, with or without sugar, syrup solids, syrup, glucose and dextrin, and/or pectin and thickener), as well as mixed egg mixtures and mixed liquid egg mixtures, cholesterol-reduced egg products and liquid products and mixtures thereof, and related products containing less than about 10% egg solids, shelled eggs, and egg components, including but not limited to cholesterol-removed egg yolks, are treated with the FFC compositions of the present invention (as described in tables 2-7 and 10). Such terms are understood by those skilled in the art and have standard meanings depending on the industry and the application being adjusted.
Example 17b
Likewise, various FFC compositions of the present invention, including but not limited to those that at least partially replace isobutyric acid with propionic acid, can be used to prepare and/or package Extended Shelf Life (ESL) liquid egg products, including but not limited to whole eggs, mixed mixes, egg yolk and egg white liquid products.
Example 17c
Likewise, various FFC compositions of the present invention may also be used in the processing of comminuted empty eggshells. It is understood in the art that using available techniques and processing equipment, the FFC composition and/or components thereof, alone or as part of another composition, can be applied (sprayed) to the vacant shell prior to further processing, e.g., into a health food product. Likewise, one or more compositions of the present invention may be applied to or added to or used in the treatment of poultry carcasses (carcas), meat, or related meat products using equipment and techniques known in the art. By extension, those skilled in the art will appreciate that the present invention may also be used with other types of animal carcasses, meats, processed meat products, and all other forms of animal meat (e.g., mammals, birds, fish, snails, clams, crustaceans, seafood, and other edible species), as described by one or more of the examples below.
Example 17d
As an extension of the previous example, the FFC composition may be added to processed health foods (e.g., herbal and spice capsules or tablets) to inhibit bacterial/fungal growth.
Example 17e
Although the preceding examples illustrate various downstream processing applications, the invention is also more broadly applicable to the field of egg and poultry production. The FFC compositions or related components of the present invention can be added to any poultry or egg production facility and/or applied to any equipment or machinery associated therewith. For example, air or surface treatment of a chicken house or growing/spawning facility can control, reduce, and/or inhibit airborne and surface deposited contaminants and subsequent microbial growth thereon.
Example 18
The FFC composition or one or more components thereof can be added to a variety of other processed foods, including foods that are water-active or support the growth of microorganisms. For example, the composition or component may be added to humus (humus), peanut butter and other such coatings (spraads), dips and mixtures. In connection with the peanut growing and processing industry, the compositions and related components of the present invention may be applied to peanuts before and after dehulling, initially washed peanuts, or related processed products (e.g., peanut butter) and/or packaging equipment and packaging materials.
Example 19
Also, the FFC compositions/components of the present invention (e.g., one or more of the compositions of tables 2-7 and 10 above, or variations of the types described therein) can be used as or added to a variety of skin care or treatment products regardless of the dosage form (e.g., lotion, ointment, cream, etc.).
Example 19a
For example, acne is often caused by one or more bacterial species that invade the pores of the skin. To further demonstrate the utility of the present invention, aqueous formulations of the propionic acid-substituted FFC compositions of the present invention can be prepared and used to treat adolescent male subjects with age-related acne. Administration every three days for three weeks significantly reduced the number and extent of acne lesions by visual observation.
Example 19b
To demonstrate another application of the present invention in the field of use and/or health care products, the FFC composition of the present invention was added (about 2 wt%) to a representative over the counter skin cream (counter skin creasertion). Referring to fig. 6, PDA plates were prepared and compared to the upper left control emulsion (without FFC component or composition), the upper right control emulsion contaminated with bacterial cells; the "treated" emulsion with FFC composition on the lower left was incubated with the treated emulsion with bacterial contamination on the lower right for one day. As shown, bacterial growth in such skin care products is inhibited by the addition of moderate concentrations (modest concentrations) of FFC compositions of the present invention.
Example 20
As such, the present invention can be used in conjunction with a range of oral hygiene, care and treatment products. The following examples demonstrate such applications of the above-described FFC compositions replaced by propionic acid, but are not limited thereto. Alternatively, a variety of other FFC compositions, or variants thereof described herein, can also be used, as described above in tables 2-7 and 10.
Example 20a
For example, in the case of one such oral care/hygiene product, a mouthwash/cleaning product is formulated using about 1% of such FFC composition. Such products are prepared by adding such FFC compositions to commercially available ready-to-use mouthwash/rinse products. Regardless of its concentration or dosage level, the FFC compositions of the present invention may also be incorporated into toothpaste/gels or related gum, mouth, oral or dental care products.
Example 20b
Lichen Planus (LP) is an autoimmune disease of the skin that can occur in the mouth or other mucous membranes. As the membrane becomes unstable, bacteria or fungi can reside in these areas and cause pain, redness, infection, bleeding, and swelling of the tissue. To reduce the cause of foreign bacteria involvement in the disease, mouthwash products were prepared comprising 1% aqueous solutions of such FFC compositions. The oral cavity of the patient is cleaned 2-3 times a day for at least 3-4 minutes each time, and then spitted out. Photographs were taken before treatment and after 3 weeks of treatment. After 3 weeks, the results show an almost complete reduction in gingival redness and, with an almost complete reduction in oral and gingival pain, the gingival and other mucosal colors return to near normal colors. Patients complain of nearly stopping pain/bleeding and maximal relief of LP compared to previous experience.
Example 20c
1% of the above FFC composition in a ready-to-use mouthwash is used to reduce plaque and treat other problems caused by bacteria associated with oral problems. After 3-4 mouthwashes per day for two consecutive months, little or no plaque formation occurs. The initial gum was recorded as red, swollen and prone to bleeding (actual recording from the dentist), now turning normal in color and no bleeding after detection using the "detector" instrument.
Example 20d
To confirm the efficacy of this FFC composition, the oral saliva obtained from the above examples was placed on one side of a nutrient agar plate, saliva without FFC from a commercial mouthwash was placed on the other side of the same plate, and saliva without mouthwash was placed on the other plate. Subsequently, saliva was incubated for 2 days. By comparison, saliva without mouthwash has a high bacterial load; FFC-free mouthwash saliva desirably has reduced bacterial load, but FFC mouthwash saliva does not have detectable bacteria.
Example 20e
In another embodiment, the oral surgeon tests the FFC composition prior to oral surgery (e.g., in the form of a 1% commercial rinse/rinse product). The patient placed untreated saliva on an agar plate (nutrient agar), rinsed with an FFC mouthwash solution and placed the saliva on another agar plate. After 2-3 days of incubation, there were no bacterial colonies on the FCC mouthwash treated plates, indicating the use to treat or inhibit dental or other oral infections before and after oral surgery.
Example 21
Cow mastitis is caused by the bacteria associated with uter. According to various non-limiting embodiments of the present invention, those FFC compositions or rhamnolipid modified FFC compositions described below may be applied to uter at the time of milking to reduce the chance of bacterial infection and contamination of the milk product.
Example 22
Various FFC compositions of the present invention can also be used to reduce microbial load on industrially/medically important biofilms (bifilms). In the latter regard, articles ranging from dental prostheses to prosthetic joints can all be treated with the FFC compositions of the present invention prior to surgical implantation.
Example 23
The FFC compositions of the present invention are useful for controlling fungal and bacterial spoilage of clothing, particularly those that come into contact with humid environments (i.e., leather, shoes, boots, belts, ties, belts). For example, 0.2ml of 1% FFC composition as described above was applied as placed on a fully wet boot. The boots were placed in an envelope to hold the resulting vapor for several hours, followed by exposure to dry air. The results showed no putrefaction and no residual musty smell after boot drying.
Example 24
The compositions of the present invention may comprise a variety of FFC components, and one skilled in the art can formulate the compositions of the present invention with an understanding of the present invention. Regardless of the end use or treatment, one or more of the FFC components of the present invention and/or related compositions can be added to a variety of antimicrobial or antifungal compositions, but are not limited thereto. Such compositions may comprise a rhamnolipid surface active component alone or in combination with such antibacterial and/or antifungal components known in the art. For the latter, such compositions may comprise syringomycins and/or pseudomycin components.
More specifically, it will be understood by those skilled in the art that the rhamnolipid component may include one or more compounds described in U.S. patent nos. 5,455,232 and 5,767,090, each of which is incorporated herein by reference in its entirety. It will also be appreciated by those skilled in the art that such rhamnolipid compounds, whether now known in the art or later isolated and/or characterized, may have the structures disclosed therein or altered structures. For example, whether synthetically derived or naturally occurring (e.g., from a pseudomonas species or strain thereof) in acid form and/or as the corresponding acid salt, such compounds may have alkyl-and/or acyl-substitution (e.g., methyl and/or acetyl, respectively, and higher homologs thereof) at one or more of the sugar hydroxyl sites. Likewise, such compounds, whether in mono-and/or dirhamnose form, may be altered by the hydrophobic moiety. As a non-limiting example, referring to fig. 7A and 7B, m and n can independently range from about 4 to about 20, whether the moiety is saturated, mono-unsaturated or poly-unsaturated, whether the hydrophobic moiety is protonated, exists as a conjugate base with any counter ion, or is otherwise derivatized. Consistent with the broader scope of the present invention, the rhamnolipids that can be used in this composition are structurally limited only by the surface active function obtained and/or the antimicrobial effect associated with the FFC composition of the present invention. Thus, those structural variants described in International publication WO 99/43334, the disclosure of which is incorporated herein by reference in its entirety, are also considered to be the subject of the present invention. See also the non-limiting rhamnolipid components/structures of figures 8-9.
Regardless of the identity of the antimicrobial or rhamnolipid, the carrier component of the present composition may comprise a fluid selected from, but not limited to, water, alcohols, oils, gases, and combinations thereof. For example, although the composition is not limited with respect to the amount or concentration (e.g., wt.%) of antimicrobial or rhamnolipid, carriers comprising water and/or alcohol may be used to facilitate desired formulation, transport, storage and/or application properties, as well as effective concentrations and resulting activities.
Such rhamnolipid surface-active components, antifungal components and/or related compositions include, but are not limited to, those described in co-pending application No.11/351,572, particularly examples 9-15 thereof, filed on 10/2/2006 and incorporated herein by reference in its entirety. Such rhamnolipid surface active components, antifungal components and/or related compositions may be added to or used in combination with one or more FFC components and/or FFC compositions of the present invention. Such antibacterial and/or antifungal components are known to those skilled in the art and are commercially available. Various rhamnolipid components and related surface active compositions are available from Jeneil Biosurfactant, LLC under the trade name Zonix.
Example 25
For example, to illustrate such rhamnolipid-related variants, a range of compositions can be prepared using one or more rhamnolipid components and one or more FFC compositions of the invention (and/or one or more FFC components thereof) for use as or in conjunction with post harvest cleaning or treatment for a variety of fruits and vegetables. In such a composition, the rhamnolipid component (e.g., as described in the above-identified' 572 application) may be present in a range of about 0.1 wt.% to about 99.9 wt.%, and the FFC composition/component (e.g., of tables 2-7 and 10 above) may be present in a range of about 99.9 wt.% to about 0.1 wt.%, but is not limited thereto. There is no tolerance limit (tolerance limit) for the aforementioned zoix rhamnolipid surfactants, with reference to applicable EPA regulations. Likewise, there are no acceptable limits to the FFC compositions/components of the present invention. Thus, foods treated with such rhamnolipid/FFC compositions can be consumed directly without washing.
Example 25a
The rhamnolipid/FFC composition can be used to wash citrus fruit as described above. One such wash/rinse composition was formulated using an 8.5% rhamnolipid solution (in water) and a 5% FFC solution (e.g., an aqueous solution of the composition of table 10). One gallon of a 95: 5(v/v) mixture was diluted to 425 gallons. The compositions are used to effectively clean and penetrate orange peel to kill bacteria and fungi on and in surfaces using procedures known in the art, or as required by applicable state or federal regulations. While effective results have been demonstrated in citrus fruits, this composition and related rhamnolipid/FFC compositions can similarly be used in conjunction with post harvest cleaning or treatment of any fruit or vegetable (such as, but not limited to: blueberries, tomatoes, grapes, onions, beets, sweet potatoes, apples, pears, pineapples, and a variety of other tropical products, such as, but not limited to, noni and cashews, and the like). Fruits/vegetables cleaned or treated using the FFC composition of this embodiment can be considered safe and sanitary for human consumption.
Example 25b
Whether or not having an added rhamnolipid component, various FFC compositions of the present invention can be used to treat various fruits and vegetables (such as, but not limited to, pears, peaches, apples, tomatoes, apricots, mangoes, etc.) before or after packaging or canning to reduce bacterial/fungal loads.
Example 26
Source of FFC component compounds: the component compounds for use in the compositions of the present invention may be obtained commercially or prepared using synthetic techniques well known or described in the literature. (see, e.g., U.S. patent No.6,911,338, which is incorporated herein by reference in its entirety).
Alternatively, as may be preferred in connection with certain embodiments, including but not limited to animal and human food and beverage items, personal care and cosmetic products and related processing and manufacturing techniques, the GRAS component compounds of the present invention and related FFC compositions may be naturally derived by fermentation techniques and are available from Jeneil Biotech, Inc, of Saukville, wi. Thus, depending on the end use or application, various compositions of the present invention may comprise various mixtures of compounds derived from bacterial fermentation, chemically synthesized compounds, and compounds of fermentative and synthetic origin.
The following examples, with reference to the above, illustrate non-limiting applications or additions of one or more compositions of the present invention, such as would be understood by one skilled in the art from a description of the present invention and various prior patents, each of which is incorporated herein by reference for the sole purpose of confirming that one skilled in the art can understand the application or addition of the present invention.
Example 27
Illustrating other embodiments, various compositions of the present invention can be formulated for use as additives to fruit beverages, as described in incorporated U.S. patent application No.6,566,349. For example, the compositions of the present invention may be combined with or added to fruit juices as a replacement for flavonoids and/or antioxidants, or may be pre-added to fruits and vegetables prior to processing to increase product shelf life. One skilled in the art will appreciate that such compositions of the' 349 patent can be modified to include one or more compositions of the present invention, the amount of which can be determined in a straightforward manner for any end use application without undue experimentation.
Example 28
The compositions of the present invention can be formulated for the preservation of tea and tea/fruit mix beverages as described in incorporated U.S. patent No.5,866,182. For example, the compositions of the present invention may be used in combination with or as a replacement for potassium sorbate and/or sodium benzoate, ascorbic acid, and dimethyl dicarbonate. It will be appreciated by those skilled in the art that such beverages of the' 182 patent (e.g., example 1 thereof) can be modified to include one or more compositions of the present invention in amounts which can be determined in a straightforward manner for any particular use without undue experimentation.
Example 29
The compositions of the present invention may be formulated for preserving and/or enhancing the antimicrobial effect of antiperspirants and deodorants, as described in incorporated U.S. patent No.5,176,903. For example, the compositions of the present invention may be used in combination with or as a replacement for parabens, imidazolidinyl urea, quaternary ammonium salt-15, benzyl alcohol, phenoxyethanol, and various other suitable preservatives (as described in examples 1-3 herein) and added to such antiperspirants and deodorants to prevent degradation, extend shelf life, and/or improve efficacy, and one skilled in the art can determine the level of one or more of the compositions in a straightforward manner without undue experimentation.
Example 30
The compositions of the present invention may also be formulated for use in antiperspirants, as described in incorporated U.S. patent No.4,548,808. For example, an effective amount of one or more compositions of the present invention can be added to a substantially anhydrous, non-alcoholic antiperspirant product as described in the' 808 patent (e.g., examples 1-6 thereof) to extend shelf life and enhance antimicrobial effectiveness, which can be readily determined by one skilled in the art without undue experimentation.
Example 31
The compositions of the present invention may also be formulated for use in animal/pet foods, such as dog foods, as described in incorporated U.S. patent No.3,119,691. One skilled in the art will appreciate that one or more compositions of the present invention can be added to low hydration (low hydration), high moisture (high texture) and rehydratable (rehydratable) dog foods, such as the product formulations described herein, to extend the shelf life of the products described in the' 691 patent, the content of such compositions being readily determined without undue experimentation.
Example 32
The compositions of the present invention can also be formulated for use in cat litter, as described in incorporated U.S. patent No.5,060,598 and U.S. patent No.4,721,059. Various adsorbent materials, including materials such as clay, alfalfa (alfalfalfa), wood chips and sawdust, and higher adsorbent materials including clay-based fillers (the '059 patent) and peat (the' 598 patent), are useful for adsorbing urine and controlling odors. One or more compositions of the present invention can be used in conjunction with (e.g., sprayed onto or incorporated into) these materials to reduce or eliminate microbial activity and control odor after use of the litter, and the amount of such compositions can be readily determined without undue experimentation.
Example 33
The compositions of the present invention may also be formulated for use in spray disinfectant applications, as described in incorporated U.S. patent No.6,250,511. The' 511 patent describes a spray bottle containing a treatment fluid comprising about 25% to 75% of at least one glycol compound, 0.2% to 60% of an antimicrobial component, about 5% to 45% of a surfactant, and optionally an effective amount of fragrances, dyes, and other additives (at column 3 thereof). For example, one or more compositions of the present invention may be used in combination with the disinfectants of the' 511 patent as a substitute for antimicrobial components, or added as an additive, and the amount of such compositions may be readily determined without undue experimentation.
Example 34
The compositions of the present invention may also be formulated for use in cleaning and/or sanitizing food and beverage processing equipment, as described in incorporated U.S. patent No. re 40,050. '050 Reissue teaches halogen dioxide compositions, and one skilled in the art can modify such formulations to replace one or more compositions of the present invention, the content of such compositions being readily determined without undue experimentation, such compositions being contacted or applied to such processing equipment using equipment and techniques as described in' 050 Reissue (as described in columns 3-4 thereof).
Example 35
The compositions of the present invention may also be formulated for wood preservation as described in incorporated U.S. patent No.4,988,576 (as well as for lignocellulose-based compositions as described in incorporated U.S. patent No.7,449,130). The' 576 patent teaches immersing wood in a solution of a preservative composition comprising at least one metal salt of a graft copolymer of a lignosulfonate, a mixture of hydroxybenzyl alcohol and a metal salt or metal salts, or a graft copolymer of a lignosulfonate, the copolymer being the reaction product of a lignosulfonate and an acrylic monomer. For example, one or more compositions of the present invention may be used alone or in combination with such preservatives as taught in the '576 patent (or the' 130 patent) (described in examples 1-4 and 1-2, respectively) to soak and preserve wood, the content of such compositions being readily determinable by one skilled in the art without undue experimentation.
Example 36
The compositions of the present invention may also be formulated for disinfecting and/or sanitizing wipes (wipes), as described in incorporated U.S. patent No.4,575,891, which teaches a pad that is partially saturated with disinfectant (as in column 2 thereof). The' 891 patent describes suitable disinfectants in the form of alcohol solutions and other preservative solutions. For example, one or more compositions of the present invention may be used alone, or in combination with such disinfectants, and incorporated into such wipe materials, the content of such compositions being readily determined and incorporated by those skilled in the art without undue experimentation.
Example 37
The compositions of the present invention may also be formulated for use with hand sanitizers, as described in incorporated U.S. patent No.6,187,327. For example, one or more compositions of the present invention may be formulated to be added to and used in conjunction with the lotion of the' 327 patent, or to replace any active ingredient in the lotion to improve antimicrobial effectiveness. The' 327 patent also discloses various other known hand sanitizing solutions (e.g., amphoteric-cationic surfactants, wetting agents, and nonionic desizing agents). However, the compositions of the present invention may be used as an alternative to, or in combination with, any active ingredient of any such hand sanitizer, and the amount of such compositions may be readily determined without undue experimentation.
Example 38
The compositions of the present invention may also be formulated for treatment of edible or agricultural seeds as described in incorporated U.S. patent No.4,581,238, which teaches contacting the seeds with a vapor having sorbate dispersed therein (e.g., column 2-5 thereof). For example, one or more compositions of the present invention may be volatilized or applied to such seeds using the techniques and devices disclosed herein, and the content of such compositions can be readily determined and incorporated without undue experimentation by those skilled in the art.
Example 39
The compositions of the present invention may also be formulated for use in preventing or inhibiting the growth of spoilage organisms, as described in incorporated U.S. patent No.4,356,204, which teaches contacting food with an effective growth inhibiting amount of ketocaproic acid (as shown at columns 2-3 thereof). One or more compositions of the present invention may be used alone or in combination with ketocaproic acid to further inhibit and/or kill spoilage organisms. Also, incorporated U.S. patent No.2,711,976 suggests the use of amino acids to increase resistance of mousse foods to spoilage organisms and staphylococcal species. Furthermore, one or more of the compositions of the present invention may be used alone or in combination with, or as a replacement for, such an amino acid. Also, incorporated U.S. patent No.2,866,819 suggests the use of sorbic acid as a preservative in foods. Furthermore, one or more of the compositions of the present invention may be used alone or in combination with, or as a replacement for, this sorbic acid. Also, incorporated U.S. patent No.2,910,368 suggests the use of EDTA and sorbic acid to increase the shelf life of vegetables. Furthermore, one or more of the compositions of the present invention may be used alone or in combination with EDTA and/or sorbic acid. In each case, such compositions of the present invention can be used in amounts readily determined by one skilled in the art without undue experimentation.
Example 40
The compositions of the present invention may also be formulated for the treatment of fruits, seeds, grains and legumes, as described in incorporated U.S. patent No.5,273,769, which teaches placing any item to be treated in a container followed by the addition of carbon dioxide and ammonia. For example, one skilled in the art will appreciate that using the devices and techniques described herein (e.g., examples 1-4), one or more compositions of the present invention can be effectively used without undue experimentation.
EXAMPLE 41
The compositions of the present invention may also be formulated for the treatment of dental and medical articles/devices and implants, the latter of which is described in more detail in incorporated U.S. patent No.6,812,217, which teaches the application of an antimicrobial polymeric film to the outer surface of an implantable medical device. For example, using the techniques described herein, one or more compositions of the present invention may also be deposited or incorporated onto such devices or articles (whether medical or dental) or polymeric films (as described in columns 5-6) to provide antimicrobial effects, the amount of such compositions being readily determined by one skilled in the art without undue experimentation.
Example 42
The compositions of the present invention may also be formulated for the treatment of fabrics as described in incorporated U.S. patent No.5,968,207, which teaches the application of triclosan ester to fabric fibers or fabrics by diffusion or soaking. For example, one or more compositions of the present invention may be formulated alone or in combination with such compounds to improve the antimicrobial properties of (synthetic, natural, or hybrid) fabrics or fibers thereof (as described in columns 2-3 of the' 207 patent), the amount of such compositions being readily determined by one skilled in the art without undue experimentation.
Example 43
The compositions of the present invention can be formulated for use in treating food processing equipment, related devices, and surfaces of food, as described in incorporated U.S. patent No.7,575,744. For example, using the techniques and apparatus described herein, one or more compositions of the present invention can be formulated and placed on equipment or food surfaces of a wide range of food processing equipment, including but not limited to snacks, poultry, citrus, peanuts, and related food processing equipment/equipment (see, e.g., column 20), to reduce or eliminate microbial activity. Such compositions can be used in amounts readily determined by those skilled in the art without undue experimentation.
Example 44
The compositions of the present invention may also be formulated for the treatment of microbial-related diseases in farm animals and livestock (i.e., mastitis, foot and mouth disease, etc.) and for inhibiting the growth of microorganisms on crops, plants, cereals and other foods, as described in incorporated U.S. patent No.7,192,575, which teaches compositions and uses including clove bud oil, eucalyptus oil, lavender oil, tea tree oil and orange oil. For example, one or more compositions of the present invention can be formulated alone or in combination with the compositions of the' 575 (e.g., examples 1-2 thereof), and the amounts of such compositions can be readily determined by one skilled in the art without undue experimentation.
Example 45
The compositions of the present invention may also be formulated for preserving foods, such as condiments, sauces, marinades, condiments, spreads (spreads), butter, margarine, milk-based foods, and the like, against microbial spoilage, as described in incorporated U.S. patent No.6,156,362, which teaches combinations of antimicrobial components. One or more compositions of the present invention can be formulated alone or in combination with the components of' 362 (as in examples 1-4 thereof), and such compositions or amounts thereof can be readily determined by one skilled in the art without undue experimentation.
Example 46
The compositions of the present invention can be formulated for incorporation into a variety of different water-based and organic-based pigments, dyes, and related surface coatings, as described in incorporated U.S. Pat. No.7,659,326 and articles cited therein (e.g., Kirk-Othmer-Paint; pp.1046-1049, Vol. 17; 1996, by Arthur A. Leman, the disclosure of which is incorporated herein by reference). For example, one or more compositions of the present invention can be formulated alone or in combination with other antimicrobial components as detailed in examples 1 and 3 of the' 326 patent, the amounts of such compositions being readily determined by one skilled in the art without undue experimentation.
Example 47
The compositions of the present invention may also be formulated for use in or incorporated into a after-shave product, as described in incorporated U.S. patent No.6,231,845. For example, one or more compositions of the present invention may be used in combination with the components described in examples 1-6 of the' 845 patent to provide antimicrobial effects to such post-shave products of the prior art. The composition can be present in an amount readily determined by one skilled in the art without undue experimentation.
Example 48
The compositions of the present invention may also be formulated for use in or added to carcasses, meat or meat products (such as mammals, birds, fish, clams, crustaceans and/or other seafood and other edible species) for treatment thereof, as described in incorporated U.S. patent No.7,507,429. For example, one or more compositions of the present invention may be formulated for use alone or in combination with other antimicrobial components for incorporation into the products described in the' 429 patent. Such compositions can be present in amounts readily determinable by one skilled in the art without undue experimentation, and the corresponding products can be used or utilized by techniques and methods described in the '429 patent or by one skilled in the art in understanding the present invention (see meat processing, spraying, soaking, and treating, and compositions and components section, as described in the' 429 patent specification).
Example 49
The compositions of the present invention can also be formulated for use in or incorporated into materials for food products (e.g., for coatings or other incorporated materials), including but not limited to snack foods, cereals, or other food components, such as the snacks and cereals and materials described in incorporated U.S. patent No.7,163,708. Without being limited to how such materials are applied, one or more compositions of the present invention may be used alone or in combination with one or more antimicrobial or preservative components of such materials, as described in the detailed description of the food and coating materials of the' 708 patent. Thus, it will be apparent to those skilled in the art that this composition can be present in amounts readily determined by those skilled in the art without undue experimentation.
Example 50
The compositions of the present invention may be formulated to be added with various edible spread compositions, including but not limited to peanut butter compositions, as described in incorporated U.S. patent No.7,498,050. For example, one skilled in the art will appreciate that one or more compositions of the present invention may be used in conjunction with the edible spread product to provide or enhance the antimicrobial effect, as described in examples 1-2 of the' 050 patent, and that such compositions may be present in amounts readily determined by one skilled in the art without undue experimentation.
Example 51
The compositions of the present invention can be formulated to be added with a variety of different pest control compositions, as described in incorporated U.S. patent No.6,720,450 (sections 2-3 of its detailed description). For example, one or more compositions of the present invention may be formulated for use alone or in combination with other pest-resistant components, as described in the' 450 patent. Likewise, one or more compositions of the present invention can be formulated as described herein with suitable carrier components for use against a variety of blood-sucking insects, including but not limited to various mosquito and crop insect pests. As described herein, the compositions of the present invention can directly contact, inhibit and/or eliminate mosquitoes, including their eggs, larvae and/or adult forms. Alternatively, the compositions of the present invention may be used and/or formulated for repellent action. However, such compositions may be present in amounts readily determinable by one skilled in the art without undue experimentation, and optionally include a surfactant component. Such surfactants may be biosurfactants. Such biosurfactants may be selected from the group consisting of mono-rhamnolipids, di-rhamnolipids and combinations thereof, but are not limited thereto.
Claims (50)
1. An antimicrobial composition comprising C of natural origin2To C5The acid component of (a); c of natural origin2To C5The acid ester component of (a); c of natural origin3To C5And C of at least two natural sources isolatable from volatile by-products of isolated cultures of Muscodor crispans grown on potato dextrose agar2To C5The composition having a pathogen activity profile different from that of isolated cultured Muscodor crispans, volatile by-products thereof or said volatile by-productsThe pathogenic activity profile of the synthetic mixture of (a).
2. The composition of claim 1, wherein the acid component is selected from isobutyric acid, propionic acid, and combinations thereof.
3. The composition of claim 1 wherein the acid ester component is selected from C4Acetic acid ester, C5Acetates and combinations thereof.
4. The composition of claim 1 comprising 8-10 of said components isolatable from volatile by-products of muscodocrispans.
5. The composition of claim 4 wherein each of said components of said composition is a volatile by-product isolatable from Muscodor crispans.
6. The composition of claim 5, wherein each of said components is a fermentation product selected from the group consisting of bacterial fermentation, yeast fermentation, and fungal fermentation.
7. The composition of claim 1 comprising a rhamnolipid component.
8. An antimicrobial composition comprising 8 to 10 components selected from C of natural origin2To C5Alcohol, C of natural origin3To C5Ketones, C of natural origin2To C5Aldehyde, C of natural origin2To C5Acids and C of natural origin2To C5An acid ester, each of said components isolatable from an isolated culture of Muscodor crispans grown on potato dextrose agar, said composition having a pathogen activity profile different from that of the isolated cultured Muscodor crispans, a volatile by-product thereof, or a synthetic mixture of said volatile by-productsEach of the components of the composition is generally considered safe for human use according to chapter 21 of the federal regulations set.
9. The composition of claim 8, wherein the component isolatable from an isolated culture of Muscodor crispans grown on potato dextrose agar is isobutyric acid.
10. The composition of claim 9 wherein at least a portion of said isobutyric acid is replaced with propionic acid.
11. The composition of claim 8, wherein the component isolatable from an isolated culture of Muscodor crispans grown on potato dextrose agar is 2-butanone.
12. The composition of claim 11, wherein at least a portion of the 2-butanone is replaced by acetic acid, propionic acid, or a combination thereof.
13. The composition of claim 8, wherein the component isolatable from an isolated culture of Muscodor crispans grown on potato dextrose agar is ethanol.
14. The composition of claim 13, wherein at least a portion of the ethanol is replaced by acetic acid.
15. The composition of claim 8 comprising a rhamnolipid component.
16. The composition of claim 8 incorporated into an article of manufacture.
17. The composition of claim 16, wherein the article is selected from the group consisting of human food, animal food, packaging products, and personal care products.
18. The composition of claim 16, wherein the article is a beverage product.
19. The composition of claim 8 applied to a plant substrate.
20. The composition of claim 19, wherein the substrate is selected from the group consisting of seeds, plant surface components, fruits and vegetables.
21. The composition of claim 20, wherein the substrate is selected from the group consisting of pre-harvest fruits and vegetables and post-harvest fruits and vegetables.
22. A method of affecting microbial activity, the method comprising providing the composition of claim 8; and contacting at least one of said microorganisms and a substrate capable of supporting microbial activity with said composition in an amount at least partially sufficient to affect microbial activity.
23. The method of claim 22, wherein the substrate is selected from the group consisting of a plant surface component, an animal substrate, and a substrate of an article of manufacture.
24. The method of claim 22, wherein said contacting is selected from direct contact with said composition and contact while said composition is at least partially volatilized.
25. The method of claim 24, wherein the contacting is direct contact with the microorganism.
26. The method of claim 25, wherein the microbial activity is abolished.
27. An article of manufacture having an antimicrobial composition applied thereto, comprising one of an article medium and an article substrate component; and an added composition of claim 1, said addition being selected from the group consisting of addition to said medium, application to the surface of said substrate component, addition within said substrate component, and combinations thereof, said composition being in an amount at least partially sufficient to affect microbial activity, and each of said components of said composition generally being considered safe for human use according to chapter 21 of the code of federal regulations.
28. The article of manufacture of claim 27 selected from the group consisting of human food, animal food, packaging products and personal care products, product processing equipment and medical devices.
29. The article of manufacture of claim 28, wherein the human food product is a beverage product.
30. The article of manufacture of claim 29, wherein the beverage product is a fruit beverage, wherein the composition is added to a medium of the beverage.
31. The article of manufacture of claim 28, wherein the medical instrument is a dental instrument.
32. The article of manufacture of claim 28, wherein the human food is meat and the composition is applied to the surface of the substrate component.
33. The article of manufacture of claim 28, wherein the human food product is a meat product and the composition is applied to the surface of the substrate component.
34. The article of manufacture of claim 28 wherein the animal food is pet food and the composition is applied to the surface of the substrate component.
35. The article of manufacture of claim 28, wherein the packaged product is a film and the composition is applied in at least one of: is applied to the surface of the matrix component and is added to the matrix component.
36. The article of manufacture of claim 28 wherein said personal care product is a skin lotion, said composition being added to said lotion medium.
37. The article of manufacture of claim 28, wherein the product processing equipment is a device for processing human food, the composition being applied to a surface of the substrate component.
38. The article of manufacture of claim 28, wherein the device is selected from the group consisting of a medical device and a medical implant, the composition being applied to the surface of the matrix component.
39. The article of manufacture of claim 38, wherein the instrument is selected from a dental appliance and a dental implant.
40. The composition of claim 1, wherein the composition is applied to a plant substrate.
41. The composition of claim 40, wherein the substrate is selected from seeds for agricultural crops.
42. The composition of claim 40, wherein the substrate is selected from plant stems and leaves.
43. The composition of claim 40, wherein the substrate is selected from the group consisting of pre-harvest fruits, vegetables and nuts, and post-harvest fruits, vegetables and nuts.
44. The composition of claim 43, wherein said substrate is selected from the group consisting of citrus fruit, potato, and peanut.
45. A synthetic non-naturally derived antimicrobial composition comprising C2To C5The acid component of (a); c2To C5The acid ester component of (a); c3To C5And at least two C's isolatable from volatile by-products of isolated cultures of Muscodor crispans grown on potato dextrose agar2To C5The composition having a pathogen activity profile different from that of isolated cultured Muscodor crispans or volatile by-products thereof.
46. The composition of claim 45, wherein the acid component is selected from isobutyric acid, propionic acid, and combinations thereof.
47. The composition of claim 45, wherein the acid ester component is selected from C4Acetic acid ester, C5Acetates and combinations thereof.
48. The composition of claim 45 comprising 8-10 of said components isolatable from volatile by-products of an isolated culture of Muscodor crispans grown on potato dextrose agar.
49. The composition of claim 48, wherein each of said components of said composition is isolatable from volatile by-products of an isolated culture of Muscodor crispans grown on potato dextrose agar.
50. The composition of claim 45 comprising a rhamnolipid component.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US21475209P | 2009-04-27 | 2009-04-27 | |
| US61/214,752 | 2009-04-27 | ||
| US25731909P | 2009-11-02 | 2009-11-02 | |
| US61/257,319 | 2009-11-02 | ||
| US31561110P | 2010-03-19 | 2010-03-19 | |
| US61/315,611 | 2010-03-19 | ||
| PCT/US2010/032587 WO2010129285A2 (en) | 2009-04-27 | 2010-04-27 | Antimicrobial compositions and related methods of use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1170634A1 HK1170634A1 (en) | 2013-03-08 |
| HK1170634B true HK1170634B (en) | 2016-04-29 |
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