Patent Application
20200163333
Much progress has been made toward identification and development of biocides for controlling various molds, plant diseases and the like. However, most commercial biocides or pesticides in use are compounds which are classified as carcinogens or are toxic to wildlife and other non-target species. For example, methyl bromide is widely used as a soil fumigant and in the post-harvest treatment of microbial infections. Human toxicity and deleterious environmental effects will ultimately result in discontinued use of methyl bromide and various other synthetic biocides/pesticides. As a result, recent efforts have been directed to the identification and development of natural or biomimetic compositions demonstrating comparable antimicrobial or pesticidal effect.
One such approach relates to endophytes and associated volatile by-products. Endophytes are defined in the art as microorganisms residing in the interstitial spaces of living plant tissue, but are generally not considered to be parasitic. In particular, endophytes found in conjunction with rain forest plants have generated considerable interest for reasons relating to the antibiotic character of their volatile by-products. Several members of the Muscodor genus (i.e., M. albus, M. roseus and M. vitigenus) have been shown to produce volatile by-products exhibiting antibiotic or insecticidal character. However, the respective by-product of each species includes various naphthalene and/or azulene derivatives. Such compounds, together with other by-product components, can be toxic or otherwise unhealthy, and the corresponding mixtures are considered unacceptable for various end use applications. Accordingly, there remains an on-going search in the art to identify natural compositions and to develop biomimetic compositions absent from such compounds, that are safe for human use and demonstrate effective antimicrobial properties.
In light of the foregoing, it is an object of the present invention to provide flavorings that have antimicrobial compositions and/or methods for their use, thereby overcoming various deficiencies and shortcomings of the prior art, including those outlined above. It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the following objects can be viewed in the alternative with respect to any one aspect of this invention.
It can be an object of the present invention to provide a Muscodor species and a volatile by-product thereof, absent naphthalene and azulene (non-GRAS compounds) related compounds, in conjunction with a methodology for the prevention, inhibition and/or eradication of microbial infection.
It can be another object of the present invention to provide a system comprising such a species or strain thereof and an associated volatile by-product in conjunction with a non-indigenous medium or substrate for use against microbial infection.
It can be another object of the present invention to provide such a system and/or related methodology for use, without limitation, in the context of human and animal food, produce, plants, plant parts, seeds, agricultural crops and other organic materials, packaging, building materials, fibers, cloth, clothing articles, and pharmaceutical and/or medical applications.
It can be another object of the present invention to provide, in the alternative or in conjunction therewith, a range of biomimetic man-made compositions demonstrating antimicrobial activity comparable to such Muscodor species.
It can be an object of the present invention to provide one or more such compositions of components edible or otherwise safe for human use and consumption.
It can be another object of the present invention to provide a system, composite or article comprising such a non-natural, biomimetic composition in conjunction with a medium or substrate for the prevention, inhibition and/or eradication of microbial infection. It can be another object of the present invention to provide such a system, composite and/or article for use, in a context of the sort described above or illustrated elsewhere herein.
It can also be an object of the present invention to provide a method for antimicrobial and/or pesticidal treatment comprising such a composition, without limitation as to medium, carrier or substrate.
Other objects, features, benefits and advantages of the present invention will be apparent from this summary and the following descriptions of certain embodiments, and will be readily apparent to those skilled in the art having knowledge of various antimicrobial compositions and related treatments. Such objects, features, benefits and advantages will be apparent from the above as taken into conjunction with the accompanying examples, data, figures and all reasonable inferences to be drawn therefrom, alone or with consideration of the references incorporated herein.
In part, the present invention can be directed to a system comprising at least one of a strain of M. crispans, a volatile by-product thereof or vapor of such a volatile by-product and a non-indigenous medium or substrate. Such media or substrates can be as described herein or as would otherwise be understood by those skilled in the art. Regardless, such a strain can be provided in the form of a biologically pure culture, optionally in conjunction with a carrier component suitable for media/substrate contact or end-use application, such a culture sufficiently viable for production of a volatile by-product. In accordance with this invention, a by-product or a modification of a by-product of M. crispans, or vapor corresponding thereto, is as compositionally described elsewhere herein.
Accordingly, the present invention can also be directed to using such a system and/or the volatile fungal by-products thereof to provide antimicrobial effect. Such a method can comprise providing a non-indigenous substrate or medium capable of supporting microbial activity or growth; and contacting such a substrate or a medium with a culture of a strain of M. crispans, a volatile by-product thereof and/or vapor from such a by-product. In certain embodiments, such contact can comprise such a strain on, about or approximate to such a medium or substrate. In certain other embodiments, a volatile by-product or modifications of a by-product of M. crispans, or a corresponding vapor, can infuse or otherwise contact such a medium or substrate.
Without limitation as to any such system or method, such a substrate can be selected from a food or produce item, a packaging component for a food or other perishable item, a fiber, clothing or clothing item, a building or construction component, a plant, plant surface, soil, garbage or refuse. Such contact can be bioactive with respect to microbial presence and/or prophylactic.
In part, the present invention can be directed to a non-naturally occurring antimicrobial composition, whether the components thereof are naturally-derived, chemically-synthesized or a combination thereof. Such a composition can comprise compounds selected from alcohol, aldehyde, ketone, acid and/or acid ester components of a biomimetic Muscodor sp. by-product composition, such a composition as can be absent fused aromatic compounds, substituted fused aromatic compounds and hydro derivatives of such compounds. In certain non-limiting embodiments, such a composition can comprise an acid component selected from acetic acid, isobutyric acid, propanoic acid and combinations thereof.
In certain embodiments, the present invention can be directed to a naturally-derived antimicrobial composition comprising a C2-about C5 acid component; a C2-about C5 ester component; and at least two C2-about C5 components isolatable from a volatile by-product of an isolated culture of Muscodor crispans, such a composition as can have a pathogen activity profile different from a pathogen activity profile of an isolated, cultured Muscodor sp., a volatile by-product thereof and/or a synthetic mixture of such a volatile by-product. Such an acid component can be selected from isobutyric acid, propanoic acid and combinations thereof. Independently, such an ester component can be selected from a C4 ester acetate, a C5 ester acetate and combinations thereof.
In certain other embodiments, such a composition can comprise propanoic acid, and a component selected from a C2-about C5 acid ester, an aldehyde and combinations thereof. In certain such embodiments, an acid ester component can be selected from acetic acid esters, isobutyric acid esters and combinations thereof. One such embodiment can consist essentially of propanoic acid and isobutyl isobutyrate. Another such embodiment can consist essentially of propanoic acid, isoamyl acetate and an aldehyde such as benzaldehyde.
Without limitation, in certain other embodiments, such a composition can comprise about 8-about 10 components otherwise isolatable from a volatile by-product of M. crispans. In certain such embodiments, each component of such a composition can be isolatable from such a volatile by-product. As such a composition can be naturally-derived, each such component can be a fermentation product, and fermentation can be selected from bacterial, yeast and/or fungal fermentations. Regardless, each such component of such a composition can be generally recognized as safe for human consumption under Chapter 21 of the United States Code of Federal Regulations and corresponding sections and/or provisions thereof.
Regardless, in certain non-limiting embodiments, such an isolatable component can be isobutyric acid. In certain such embodiments, propanoic acid can be at least in part substituted for isobutyric acid. In such or other non-limiting embodiments, such an isolatable component can be 2-butanone. In certain such embodiments, acetic acid, propanoic acid or a combination thereof can at least in part be substituted for 2-butanone. In such or yet other non-limiting embodiments, such an isolatable component can be ethanol. In certain such embodiments, acetic acid can be at least in part substituted for ethanol. Regardless of the identity or amount of any such acid component, ester component and/or isolatable component, such a naturally-derived composition can comprise a surfactant component. In certain such embodiments, a biosurfactant can be incorporated therewith. Without limitation, a biosurfactant can be a rhamnolipid component selected from a monorhamnolipid, a dirhamnolipid and combinations thereof.
Alternatively, the present invention can be directed to a synthetic, non-naturally derived antimicrobial composition. Such a composition can comprise a C2-about C5 acid component; a C2-about C5 ester component; and at least two C2-about C5 components isolatable from a volatile by-product of an isolated culture of Muscodor crispans, such a composition as can have a pathogen activity profile different from a pathogen activity profile of an isolated, cultured Muscodor sp. or a volatile by-product thereof. Such acid, ester and/or isolatable components can be as described above or illustrated elsewhere herein. Regardless, such an antimicrobial composition can comprise a surfactant component. In certain such non-limiting embodiments, such a surfactant can be a rhamnolipid component selected from a monorhamnolipid, a dirhamnolipid and combinations thereof.
In part, the present invention can be directed to a biomimetic, antimicrobial composition comprising a liquid mixture of compounds selected from C2 to about C5 alcohols, aldehydes, ketones, acids and acid esters and combinations and sub-combinations thereof, such a composition not isolated from Muscodor sp. As discussed elsewhere herein, such a liquid mixture can be volatile at room and/or ambient temperatures. With respect to such a composition and the compounds thereof, the term “about” can mean, as would be understood by those skilled in the art, carbon and/or methylene homologs with corresponding molecular weight and/or structural isomerism limited only by mixture with one or more other components, compounds and at least partial room/ambient temperature volatility of the resulting composition. With respect to certain non-limiting embodiments, such a composition can comprise alcohol, aldehyde, ketone, acid and acid ester compounds selected from components of a biomimetic M. crispans by-product composition, of the sort described below. Such a composition can comprise compounds chemically synthesized, compounds isolated from bacterial fermentation and combinations of such compounds. In certain such embodiments, such a composition can comprise an acid component selected from acetic acid, isobutyric acid, propanoic acid and combinations thereof.
In part, the present invention can also be directed to a non-naturally-occurring, whether naturally-derived and/or chemically-synthesized, antimicrobial composition comprising compounds selected from C2 to about C5 alcohols, aldehydes, ketones, acids and acid esters and combinations and sub-combinations of such compounds, such selected compounds generally recognized as safe (“GRAS”) for human consumption, such designation as provided in Chapter 21 of the United States Code of Federal Regulations and corresponding sections and/or provisions thereof. In certain non-limiting embodiments, such compounds can be selected from alcohol, ketone, acid and/or acid ester components of a biomimetic M. crispans by-product composition. In certain embodiments, a microbe activity/mortality profile thereof differs from that of either M. crispans or M. albus, a volatile by-product thereof and/or corresponding synthetic by-product compositions thereof. Regardless, in certain such embodiments, such a composition can comprise an acid component selected from acetic acid, isobutyric acid, propanoic acid and combinations thereof.
In part, the present invention can comprise a composition comprising a composition of this invention; and a surfactant component, such a surfactant component alone or as can be incorporated into a carrier component. In certain embodiments, such a surfactant can be a biosurfactant, such a biosurfactant as can be a rhamnolipid component selected from a monorhamnolipid, a dirhamnolipid and combinations thereof.
In part, the present invention can also be directed to a system or composite comprising an inventive composition and a substrate or medium component. Such a composition can be as described above or illustrated elsewhere herein. Without limitation, a substrate can be selected from a food or produce item, a packaging component (e.g., a film or wrapper) for a food or other perishable item, a fiber, cloth or clothing item, a building or construction component, a human tissue, a plant, plant surface, soil, and garbage or refuse. In certain embodiments, such a composition, whether liquid or gaseous, can be incorporated or otherwise in contact with such a medium, substrate or substrate surface.
In part, the present invention can be directed to an article of manufacture, such an article as can comprise a solid carrier component and a volatile antimicrobial composition absorbed in, adsorbed on, coupled to or otherwise incorporated therewith. Such an antimicrobial composition can comprise propanoic acid; and a component selected from a C2-about C5 acid ester, an aldehyde and combinations thereof. In certain embodiments, such an acid ester can be selected from acetic acid esters, isobutyric acid esters and combinations thereof; and/or an aldehyde component can be selected from a C2-about C8 aldehyde component. In certain such embodiments, an acid ester can be isobutyl isobutyrate; or such an aldehyde component can be benzaldehyde. Regardless, such an antimicrobial composition can be incorporated with a carrier component comprising a clay. In certain such embodiments, such a carrier component can comprise a bentonite clay. Regardless, such a composition can comprise one or more optional components or adjuvants, including but not limited to a rhamnolipid component.
Alternatively, such an article of manufacture can be considered with an incorporated antimicrobial composition as can comprise propanoic acid and a C2-about C5 acid ester comprising at least one of an alkylcarbonyl group, RC(O)—, wherein R comprises an isopropyl, (CH3)2CH—, moiety, and an alkoxy group, —OR′, wherein R′ comprises an isopropyl, —CH(CH3)2, moiety. In certain embodiments, such an acid ester can be selected from isoamyl acetate, isobutyl isobutyrate and a combination thereof. In certain such embodiments, such an antimicrobial composition can consist of propanoic acid and isobutyl isobutyrate. In certain other such embodiments, such a composition can consist of propanoic acid, isoamyl acetate and benzaldehyde. Regardless, such a solid carrier component can comprise a clay.
Accordingly, such an article can comprise a solid carrier component comprising a bentonite clay, and an antimicrobial composition incorporated therewith. Without limitation, such an antimicrobial composition can be selected from a composition consisting essentially of propanoic acid and isobutyl isobutyrate; and a composition consisting essentially of propanoic acid, isoamyl acetate and benzaldehyde.
In part, the present invention can also be directed to an article of manufacture comprising granules of a solid carrier component and an antimicrobial composition incorporated therewith. Such a composition can comprise a C2-about C5 acid component; at least one C2-about C5 component isolatable from a volatile by-product of an isolated culture of Muscodor crispans grown on potato dextrose agar; and a component selected from at least one C2-about C5 acid ester, an aldehyde and combinations thereof. As relates to such an article, such composition-incorporated solid carrier component granules can be provided in a vapor-permeable enclosure.
In certain embodiments, such solid carrier and antimicrobial compositions can be as discussed above or illustrated elsewhere herein. In certain such embodiments, such an antimicrobial composition can be about 0.01 wt. % to about 10.0 wt. % of such an article. In certain such embodiments, such a composition can be about 0.20 wt. % to about 10.0 wt. %. In yet other embodiments, such a composition can be about 1.0 wt. % to about 3 wt. % of such an article. Without limitation as to solid carrier component or antimicrobial composition incorporated therewith, such an enclosure can comprise a woven mesh and/or non-woven material as can be configured as a flexible bag or pouch. Regardless, such an article of manufacture can be provided in a container with a perishable food item.
Accordingly, this invention can also be directed toward a method of microbial or insect treatment, prevention, inhibition, eradication and/or to otherwise affect microbial or insect activity. Such a method can comprise providing a composition of this invention, including but not limited to one or more compositions of the sort illustrated herein; and contacting a microbe or insect or an article/substrate capable of supporting microbial or insect activity with such a composition in an amount at least partially sufficient to affect microbial or insect activity. Such a microbe (e.g., a fungus, bacterium or virus) or insect can be in a medium, on or about a surface of a substrate of the sort discussed above. Accordingly, such contact can be direct and/or upon volatilization of such a composition. Regardless, such treatment can be active with respect to microbial or insect presence and/or prophylactic. As illustrated elsewhere herein, treatment can be considered in the context of microbial or insect death and/or inhibited growth or activity.
In part, the present invention can also be directed to a method of affecting microbial activity. With respect to such a method, the present invention can comprise providing an article of the sort described above or illustrated elsewhere herein; and contacting the vapor of an antimicrobial composition of such an article with a microbe and/or a food item capable of supporting microbial activity, such a composition as can be in an amount sufficient to affect microbial activity. In certain embodiments, such a food item can comprise post-harvest produce. Together with an article of this invention, such produce as can be optionally introduced to a container at a point along a produce supply chain. Without limitation, produce introduction can be at a point of harvest, a point of processing, a point of wholesale distribution, a point of retail sale, and combinations thereof. Regardless of produce or point of introduction, such an antimicrobial composition can be as described above or illustrated elsewhere herein.
As discussed below and illustrated by several non-limiting examples, this invention can comprise one or more acid salts. Accordingly, in part, the present invention can be directed to one or more compositions comprising a propanoic acid component; and a component selected from a C4-about C6 acid salt component, a C2-about C5 acid ester component, a C2-about C8 aldehyde component and combinations thereof. In certain embodiments, such an acid salt component can be selected from salts of isobutyric acid, salts of citric acid and combinations thereof. In certain such embodiments, such an acid salt component can be selected from salts of isobutyric acid and combinations thereof. Without limitation, such a salt can be selected from potassium and ammonium salts of butyric acid. Regardless of the presence of an acid salt component, such an ester component can be selected from esters of a C4 acid and combinations thereof. Likewise, regardless of the presence of an acid salt and/or ester component, such an aldehyde component can be benzaldehyde. In certain other embodiments, regardless of the presence of an acid salt component, ester component and/or aldehyde component, such a composition can comprise a C2-about C6 acid component in addition to propanoic acid. In certain such embodiments, such an additional acid component can be selected from acetic acid, isobutyric acid, citric acid, and combinations thereof.
Without limitation, such a composition can comprise propanoic acid and at least one C4 acid salt. In certain embodiments, such an acid salt can be selected from potassium and ammonium salts of isobutyric acid. In certain such embodiments, such a composition can comprise an acid component in addition to propanoic acid. Such an additional acid component as can be selected from acetic acid, isobutyric acid, citric acid and combinations thereof. In certain other embodiments, such a composition can comprise propanoic acid and at least one C2-about C5 acid ester component. Certain such embodiments can comprise at least one C4 acid ester. Regardless, such a composition can comprise an acid component in addition to propanoic acid, such an additional acid component as can be selected from acetic acid, isobutyric acid, citric acid and combinations thereof.
In part, the present invention can also be directed to compositions comprising propanoic acid and at least one C4-about C6 acid salt component. In certain embodiments, such an acid salt component can be selected from salts of isobutyric acid, salts of citric acid and combinations thereof. In certain such embodiments, such an acid salt can be a salt of isobutyric acid. Without limitation, such an acid salt component can be selected from potassium and ammonium salts of isobutyric acid, and combinations thereof. Regardless of the identity of such an acid salt component, such a composition can comprise an acid component in addition to propanoic acid, such an additional acid component as can be selected from C2-about C6 acids and combinations thereof. In certain embodiments, such an additional acid component can be selected from acetic acid, isobutyric acid, citric acid and combinations thereof. Without limitation, such a composition can be selected from a composition consisting essentially of propanoic acid and a salt of isobutyric acid; and a composition consisting essentially of propanoic acid, a salt of isobutyric acid, and at least one of acetic acid and citric acid.
As discussed above and illustrated below, such compositions can be incorporated into an article of manufacture. Accordingly, in part, the present invention can be directed to an article of manufacture comprising one or more compositions, of the sort discussed above, comprising propanoic acid. In certain embodiments, such an article can be selected from a human food product, an animal food product, an animal care product, a packaging product and a solid carrier component. Without limitation, a solid carrier component can comprise a clay. In certain other embodiments, such a human food product can be selected from processed foods. Various such articles are illustrated below, including but not limited to cheese and related dairy products.
In accordance with certain embodiments of this invention, compositions comprising certain food and flavor compounds (FFCs) are especially inhibitory and/or lethal to certain pathogenic fungi, bacteria and other microbes of agricultural, medicinal, or commercial or industrial concern. Such compositions can be distinguished over any previous mixture containing biologically derived compounds: for instance, the present compositions do not contain any naphthalene or azulene (non-GRAS compounds) derived substances. Conversely, such compositions can comprise a mixture of organic compounds, each of which otherwise considered (i.e., GRAS) a food or flavoring substance.
The present invention demonstrates the nature of such compositions, their preparation and application to various items (e.g., without limitation, food, fibers, implements and construction surfaces) to preserve their integrity and prevent destruction by various fungi (molds and other microorganisms). Such compositions can also be applied to building structures, plant parts and even clothing items for their preservation. Further, as demonstrated below, such a composition can negatively affect Mycobacterium tuberculosis—the microorganism that causes tuberculosis—including at least 3 strains that are otherwise drug resistant.
As illustrated by several non-limiting embodiments, this invention relates to the use of a new species of Muscodor and/or its volatile by-products and the development of non-natural, laboratory-prepared, biomimetic compositions comprising common food and flavor compounds that, when incorporated into various media, applied to surfaces, or introduced to an atmosphere, space or volume, bring about a decontamination of the desired surface medium or volume of otherwise unsightly, harmful, and/or pathogenic microorganisms including plant fungi and the causal agent of tuberculosis. The invention has extremely important implications and applications to modern agriculture, human medicine, food sciences, and industry. The compositions of this invention are not obvious as having antimicrobial properties given the fact that no one individual ingredient, in and of itself, is biologically active. A synergistic combination of component ingredients manifests the full potential antimicrobial activity.
With respect to the use of such a Muscodor species, a volatile by-product thereof or a non-naturally-occurring biomimetic composition comprising FFCs, contact can be direct or by exposure to a vapor associated with such a species, by-product of biomimetic composition. As illustrated below, in the context of certain embodiments, while vapor exposure can inhibit growth, direct microbial contact may be required for bacterial or fungal death.
Regardless of mode of contact, the compositions of this invention can be laboratory-made, comprising chemically-synthesized components, naturally-derived components or a combination of such synthetic and natural components. Regardless, such compositions can be biomimetic with respect to the effect of a Muscodor by-product on a particular bacterial or fungal species. Alternatively, such a composition, by relative concentration or selection of any one or more FFC component thereof, can demonstrate varied or enhanced antimicrobial activity, as compared to a Muscodor fungal by-product.
In certain such embodiments, such a composition can be on, or as can be applied to, a substrate or medium comprising a proteinaceous or cellulosic component which can, is capable of or does support microbe growth. Without limitation, certain embodiments can comprise plants, plant components (e.g., roots, stems, leaves or foliage, produce and the like) and any originating shoots or seeds. In particular, without limitation, such compositions can be on any plant produce, whether termed a fruit, vegetable, tuber, flower, seed or nut, whether before or post-harvest. Certain such plants and/or produce therefrom are recognized in the art, alone or collectively, as agricultural crops. Accordingly, in certain embodiments, a composition of this invention can be on or applied to such a crop at any time during development, pre-harvest and/or post-harvest. Likewise, a composition of this invention can be applied to or incorporated into a beverage, food (e.g., human, pet and/or animal) product or article of manufacture which can, is capable of or does support microbe growth.
In certain other embodiments of this invention, such a composition can be on, or as can be applied to, a substrate or surface supporting or supportive of microbe (e.g., yeast and/or fungi bacteria and/or virus) growth. Accordingly, such a substrate or surface can comprise any material which can, is capable of or does support microbe growth. Such substrates include but are not limited to wood, ceramics, porcelain, stone, plaster, drywall, cement, fabrics, leather, plastics and the like.
In certain other embodiments, various compositions of this invention can be on, in contact with, or as applied or administered to a substrate or surface comprising mammalian or human tissue, including but not limited to nails, hair, teeth or mouth, skin and other cellular material, in the context of a pharmaceutical or personal care or hygiene formulation for the treatment or prevention of microbial growth or infection. Representative compositions are described, below, in terms at least in part applicable to one or more other embodiments.
An endophytic fungus was recovered from inside the tissues of a wild pineapple plant (Ananas ananassoides) growing in the Bolivian Amazon. Ultimately, it was shown to produce a mixture of volatile compounds having antimicrobial activities. Using molecular techniques, the fungus was found to possess sequence similarities to members of the Muscodor genus. These fungi are known to produce volatile organic compounds that can act as anti-microbials which are effective against both human and plant pathogens. Members of the Muscodor species have been identified utilizing methods such as Phylogenetic Character mapping employing 18S rDNA plus ITS-5.8S rDNA sequence analyses. The sequences found in the present fungus and other Muscodor spp. were BLAST searched in GenBank, and compared to other fungi (Bruns et al., 1991; Reynolds and Taylor 1993; Mitchell et al., 1995; Guarro et al., 1999; Taylor et al., 1999). Ultimately it was determined that these isolates are related to Xylaria (Worapong et al., 2001a&b). All isolated taxa that belong to Muscodor have similar characteristics, such as growing relatively slowly, possessing a felt-like mycelium, producing biologically active volatile compounds, and causing no harm to the plants in which they originally resided. Finally, they each share closely similar rDNA sequences (Ezra et al., 2004).
Although the present fungus shared all of the same common features mentioned above, there were a number of different aspects to the taxon which distinguished it from all other Muscodor spp. and isolates. As illustrated more fully in the following examples, these unique characteristics support establishment of the present fungus as a new species. The name proposed for this novel endophytic fungus is Muscodor crispans.
As analyzed by GC/MS, the isolated fungus produced alcohols, esters and small molecular weight acids, in the gas phase, when grown on potato dextrose agar (PDA). As shown in Table 1, below, such compounds include propanoic acid, 2-methyl; 1-butanol, 3-methyl, acetate; 1-butanol, and ethanol. Neither naphthalene nor azulene derivatives (non-GRAS compounds) were produced by this organism when grown on PDA, distinguishing it from all other Muscodor spp. studied thus far. The odor produced by the fungus becomes noticeable after about 1 week and seems to increase with time up to and including at least three weeks. As illustrated below, the volatiles of this fungus possess inhibitory and lethal bioactivity against a number of plant and human pathogens using the standard bioassay technique (Strobel et al., 2001).
As discussed above, the present invention includes use of M. crispans and/or a volatile by-product thereof in conjunction with a non-indigenous medium, substrate and/or volume for antimicrobial effect. Such use and/or applications can be as described herein or as would otherwise be understood by those skilled in the art, including but not limited to use and application of the sort described in U.S. Pat. No. 6,911,338, the entirety of which is incorporated by reference.
Alternatively, a wide range of natural and synthetic biomimetic compositions can be used with comparable or enhanced effect or, as evidenced by one or more embodiments, to provide results heretofor not available through use of either the fungus or its volatile by-product. As a departure from the prior art and the by-product of M. crispans, such antimicrobial compositions can comprise food and flavor compounds generally recognized as safe for human use and consumption. Representative thereof, several non-limiting biomimetic compositions are provided in Tables 2-7, below. Various other compositions can comprise combinations of compounds selected from any one or more of Tables 2-7. (See, e.g., examples 52-56.) Alternatively, any such composition can comprise a component compound in addition to or as replacement for any compound listed, to enhance volatility or modify any other end-use or performance property. In certain such compositions, such a replacement or additional compound can have a GRAS designation and/or be so designated at levels utilized—such compositions as can be considered essentially free of any component or material that would not be generally recognized as safe (GRAS) under the applicable United States Code of Federal Regulations. Such compositions can, alternatively, include a component found in a volatile by-product of M. crispans and/or not in a volatile by-product of another Muscodor sp.
Each such compound can be provided within an effective concentration or percentage range and is either commercially available or can be prepared by those skilled in the art. With regard to the latter, fermentation techniques can be used to naturally prepare and isolate such compounds. Alternatively, such compounds can be chemically synthesized. With respect to several non-limiting embodiments of this invention, each compound of Tables 2-7 can be obtained as a fermentation product, such products and corresponding compositions as are available under the Flavorzon trademark from Jeneil Biotech, Inc. of Saukville, Wis.
With respect to any FFC composition of this invention, it is contemplated that any compound component thereof—including any compound component described referenced or inferred herein, such as but not limited to any component in Tables 1-7 and 10 and structural isomers and/or carbon and methylene homologs thereof—can be present in an amount or a range separate and apart from any other compositional component. Accordingly, without limitation, each such compound component can be present in an amount of or a 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 4.2 wt. %, about 4.3 wt. %, or about 4.4 wt. % . . . or/to 5.0 wt. %, about 5.1 wt. %, about 5.2 wt. %, about 5.3 wt. %, or about 5.4 wt. % . . . or/to about 6.0 wt. %, about 6.1 wt. %, about 6.2 wt. %, about 6.3 wt. %, or about 6.4 wt. % . . . or/to about 7.0 wt. %, about 7.1 wt. %, about 7.2 wt. %, about 7.3 wt. %, or about 7.4 wt. % . . . or/to about 8.0 wt. %, about 8.1 wt. %, about 8.2 wt. %, about 8.3 wt. %, or about 8.4 wt. % . . . or/to about 9.0 wt. %, about 9.1 wt. %, about 9.2 wt. %, about 9.3 wt. %, or about 9.4 wt. % . . . or/to about 10.0 wt. %; and or/to about 10.1 wt. % . . . or/to about 20.0 wt. %, in accordance with such incremental variation; or/to about 20.1 wt. % . . . or/to about 30.0 wt. %, in accordance with such incremental variation; or/to about 30.1 wt. % . . . or/to about 40.0 wt. %, in accordance with such incremental variation; or/to about 40.1 wt. % . . . or/to about 50.0 wt. %, in accordance with such incremental variation; or/to about 50.1 wt. % . . . or/to about 60.0 wt. %, in accordance with such incremental variation; or/to about 60.1 wt. % . . . or/to about 70.0 wt. %, in accordance with such incremental variation; or/to about 70.1 wt. % . . . or/to about 80.0 wt. %, in accordance with such incremental variation; or/to about 80.1 wt. % . . . or/to about 90.0 wt. %, in accordance with such incremental variation; or/to about 90.1 wt. % . . . or/to about 99.9 wt. % (or more), in accordance with such incremental variation. Likewise, without limitation, any composition of this invention—regardless of identity or amount of any particular compound component or combination—can be present in amount (wt. %) or a wt. % range incrementally variable, as described above, from 0.1 wt. % to 99.9 wt. % of any composition or medium (e.g., within any range from about 0.1 wt. % to about 1.0 wt. %, about 2.0 wt. %, about 4.0 wt. % or to about 10.0 wt. %) therein incorporated or article or substrate thereon applied.
Unless otherwise indicated, all numbers expressing amounts, concentrations or quantities of components or ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances 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. At the very least, and not as an attempt to limit application of the doctrine of equivalents to the scope of claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and the parameters setting forth the broad scope of this invention are approximations, the numerical values set forth and the examples are reported as precisely as possible. Any numerical value, however, may inherently contain a certain error resulting from the standard deviation found in a respective testing measurement.
The compositions and methods of this invention can suitably comprise, consist of or consist essentially of any compound component or amount/concentration thereof disclosed, referenced or inferred herein—including but not limited to any compound component in Tables 1-7 and 10, together with any structural isomers thereof, carbon and/or methylene homologs of any such alcohol component, aldehyde component, ketone component, acid component and/or ester component, whether the acid-derived and/or alcohol-derived moiety thereof. Regardless of amount/concentration, each such compound component or moiety/substituent thereof is compositionally distinguishable, characteristically contrasted and can be used in conjunction with the present compositions and methods separate and apart from another such component amount/concentration or another compound component (or moiety/substituent) or amount/concentration. Accordingly, it should be understood that the inventive compositions and/or methods, as illustratively disclosed herein, can be claimed, practiced or utilized with change in amount or concentration in the absence of any one component compound (or moiety and/or substituent thereof), such compound (or moiety/substituent thereof) or amount/concentration thereof which may or may not be specifically disclosed, referenced or inferred herein, the change or absence of which may or may not be specifically disclosed, referenced or inferred herein.
In preferred embodiments, a biologically effective composition of such FFCs (prepared as a liquid mixture) is readily volatilized at room temperature and diffuses throughout an enclosed space to effectively inhibit and/or kill unwanted contaminating fungi (molds) on surfaces that are desired to be free of such harmful microbes. The mixture maybe applied as a spray (e.g., can with ingredients under pressure) or simply placed in a container and allowed to evaporate in the closed container or sealed bag.
Regardless, the FFC compositions of this invention can be incorporated into a variety of end-use compositions, limited only by application. Such compositions include but are not limited to those directed to human/animal food or nutrient, personal hygiene, healthcare, agricultural, industrial, residential, medical and consumer applications. In certain non-limiting embodiments, an FFC composition and/or component(s) thereof can be present at about 0.1 wt. % or less to about 99.9 wt. % or more of a particular end-use composition. Such level of incorporation is limited only by desired antimicrobial effect and/or formulation considerations.
The present FFC compositions, under effective dose levels, are effective in killing many plant pathogens, fungi that can cause food spoilage, microbes that can cause major human diseases and microbes that can foul work surfaces, homes and other buildings. A non-exclusive list of such applications is below:
1. For treatment of cheeses in storage or in preparation to control unsightly mold contamination of surfaces and eventual spoilage of the cheese blocks.
2. For treatment of various plant parts in storage including roots, tubers, stems, seeds and other organs that may be eventually used for food preparation of for planting and re-vegetation or agricultural purposes.
3. For use in decontaminating buildings that may either have moldy surfaces or be infested to a point that a mold problem may develop.
4. For use in the preservation of garbage whilst it is in shipment over long sea hauls from one port to another for eventual fermentation into energy related products.
5. For the decontamination of soils that may harbor microbes that are potential plant pathogens.
6. For the treatment of patients with tuberculosis and other mycobacterium infections.
7. For treatment to control nasal infections and to clear nasal passage ways.
8. For combining with specifically designed polymers that can be used to wrap and thus preserve materials including foods, fibers and other items for longer term safe storage.
More generally, the compositions of this invention can be used to inhibit the growth of or kill an organism selected from the group consisting of a fungus, a bacterium, a microorganism and a range of other microbes or pests. Using methods well known to those of skill in the art, such a composition is contacted with the organism in an amount at least partially effective to kill or inhibit the growth of the organism. Alternatively, it can be used to treat human or animal waste, e.g., as a component of a waste water or solid management or treatment. Such compositions also are useful to decontaminate human and animal waste, e.g., decrease or remove bacterial and fungal contamination. Yet further, such a composition can be used to treat or prevent mold on building materials and in buildings by contacting the building, the building materials, or the spaces between the building materials with an effective amount thereof or vapors therefrom. For the purpose of illustration only, an effective amount of such a composition can be used alone or in combination with other fumigants or active agents in a room or alternatively, during whole building fumigations.
When used in agricultural applications, the invention provides a method for treating or protecting fruit, seeds, plants or the soil surrounding the plants from an infestation by an organism such as a fungus or a bacterium, by contacting the microorganism with an effective amount of one or more compositions of the sort described herein.
As discussed above, the present invention provides a method of preventing, treating, inhibiting and killing a bacterial, fungal, viral and/or other microbial infection. Such a method can comprise administering to an article, animal/mammal or plant substrate, having such an infection or growth or capable of supporting such an infection or growth, an effective amount of an inventive composition—alone or as can be incorporated into a composition or formulation. Accordingly, the present invention provides one or more compositions for pharmaceutical, personal (e.g., without limitation, cosmetic), industrial and/or agricultural use.
Microbial treatment can be achieved by contacting a bacterium, fungus, virus and/or other microbe with an effective amount of an inventive composition. Contacting may take place in vitro or in vivo. “Contacting” means that such a composition of this invention and such a microbe are brought together in a manner sufficient to prevent, inhibit and/or eliminate microbial infection and/or growth. Amounts of such a composition effective for such treatment may be determined empirically, and making such determinations is within the skill in the art. Inhibition includes both reduction and elimination of microbial growth/activity.
Compositions of this invention may be administered to or contacted with a human, animal or plant, 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, as by powders, granules, liquids, sprays, ointments, lotions or creams. Accordingly, compositions of the invention can comprise the respective component compounds in admixture with one or more acceptable carriers and, optionally, with one or more other compounds or other materials. Such a carrier 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 inventive compositions can be formulated to provide acceptable concentrations or dosage forms by conventional methods known to those of skill in the art. The amount or concentration of any such composition or component thereof, with or without a carrier, will vary depending upon the target microbe/substrate/article being treated, the particular mode of administration/delivery and all of the other factors described above. The amount combined with a carrier material will generally be that amount of such a composition providing the lowest or a minimal concentration effective to produce a desired antimicrobial effect.
The relative amounts or concentrations of an FFC composition and another optional component in the compositions of the present invention can vary widely within effective ranges, as demonstrated in the examples that follow. The concentrations and/or doses utilized are preferably selected to achieve an enhanced or increased activity over individual prior art components alone and/or to maximize the activity of the composition at the lowest effective component concentration(s). Accordingly, the weight ratios and/or percent concentrations yielding such enhanced activity depend not only on the specific FCC composition utilized, but on the specific end-use application of the composition including, but not limited to, climate, soil composition, nature of the substrate, article and/or microbial host to be treated and/or potential exposure to a particular microbe.
Methods of preparing formulations or compositions include the step of bringing a composition of this invention, or one or more component compounds, into association with a carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by bringing such a composition/component into association with a carrier (e.g., a liquid or finely divided solid carriers) and, if desired, shaping the product.
Formulations relating to the invention, whether a composition of this invention or any article of manufacture incorporating such a composition, may be in the form of capsules, cachets, pills, tablets, powders, granules, paste or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as washes (e.g., mists, spray or mouth) and the like, each containing a predetermined amount of an inventive composition or components thereof.
In other solid such formulations (e.g., capsules, tablets, pills, dragees, powders, granules and the like), a composition of this invention can be mixed with one or more other active ingredients and/or acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethyl-cellulose, 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, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol 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) coloring agents. 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 sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient(s) moistened with an inert liquid diluent.
The tablets, and other solid forms of such compositions or articles incorporating such compositions, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient(s) therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient(s) can also be in microencapsulated form.
Liquid forms for use or administration of this invention include pharmaceutically- or otherwise-acceptable emulsions, mixtures, microemulsions, solutions (including those in distilled or purified water), suspensions, mists, syrups and elixirs. In addition to an inventive composition or compound component(s) thereof, a liquid form may contain inert or other diluents commonly used in the art, such as, for example, 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.
Besides inert diluents, such compositions and/or related articles can also include adjuvants such as but not limited to wetting agents, emulsifying and suspending agents (e.g., sticker and spreader agents for agricultural application), coloring, perfuming and one or more other preservative agents. Suspensions can comprise suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.
Formulations of compositions of this invention and/or articles or products incorporating such inventive compositions for substrate or topical (e.g., in the context of a personal care or hygiene product) administration/delivery of this invention, include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. Such ointments, pastes, creams and gels may contain, in addition to an inventive composition of this 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 silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as volatile unsubstituted hydrocarbons, such as butane and propane, or be delivered under positive air pressure.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the compositions of the 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. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
Depot forms of articles or products incorporating a composition of this invention can be made by forming microencapsule matrices of an active ingredient(s) in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the active ingredient(s) to polymer, and the nature of the particular polymer employed, the rate of release of the active ingredient(s) can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the active ingredient(s) in liposomes or microemulsions which are compatible with body tissue.
Further, the compositions of the present invention and/or articles or products incorporating such a composition can comprise additional chemical and/or biological, multi-site and/or single site antimycotic or antifungal, antibacterial and antimicrobial agents, of a similar and/or different modes of action, as will be well known to those skilled in the art. Such agents can include, but are not limited to, potassium bicarbonate, silica, copper or sulfur-based compounds and/or botanical oils (e.g., neem oil). Further, such agents can 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 nikkomycins; other chitin inhibitors, elongation factor inhibitors, such as sordarin and analogs thereof; inhibitors of mitochondrial respiration, inhibitors of sterol biosynthesis and/or any other fungicidal or biocidal composition known to those skilled in the art suitable for treating or preventing yeast or fungal, bacterial, viral and/or other microbial infections of plants, other substrates, animals and/or humans, or as can be found on or in any article of manufacture.
In certain embodiments, articles or products incorporating the compositions of the present invention can also include one or more preservative components known in the art, including but not limited to, sorbic or benzoic acid; the sodium, potassium, calcium and ammonium salts of benzoic, sorbic, hydroxymethyl glycinic, and propionic acid; and methyl, ethyl, propyl and butyl paraben and combinations thereof.
The compositions of this invention may contain a compound comprising an acidic or basic functional group 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 a compound with a suitable acid or base. Suitable bases include the hydroxide, carbonate or bicarbonate of such an acceptable metal cation, ammonia, or such an acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the 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 the hydrobromide, hydrochloride, sulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthalate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like.
The compositions of the present invention can be used as aqueous dispersions or emulsions and are available in the form of a concentrate containing a high proportion of an FFC (with or without a surfactant) composition, as can be diluted (e.g., water or another fluid component) before use. Emulsifiable concentrates or emulsions may be prepared by dissolving a composition of the present invention, together with any other desired active ingredient, in a solvent optionally containing a wetting or emulsifying agent and then 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 storage for prolonged periods and after such storage be capable of dilution with water in order to form aqueous preparations which remain homogeneous for a sufficient time to enable them to be applied by conventional spray equipment.
Depending on the type of end-use application, articles or products incorporating compositions of the present invention may also comprise any other required components including, but not limited to, solid or liquid carriers to facilitate application, surfactants including biosurfactants, protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, stabilizers, sequestrants, texturing agents, flavoring agents (e.g., for post-harvest or processed food/beverage applications), sugars, colorants, etc., as will be well known to those skilled in the art.
For example, such compositions and/or related articles or products can be used for agricultural purposes and formulated with such a carrier or diluent. The compositions can be applied, formulated or unformulated, directly to the foliage of a plant, to seeds or to other medium in which plants are growing or are to be planted, or they can be sprayed on, dusted on or applied as a cream or paste formulation, or they can be applied as a vapor or as slow release granules. Application can be to, or proximate to, any part of the plant including the foliage, stems, branches or roots, or to soil surrounding the roots, fruit or vegetable (pre- or post-harvest) or to the seed before it is planted, or to the soil generally, to irrigation water or to hydroponic culture systems. The inventive compositions can also be injected into plants or sprayed onto vegetation (including fruits and vegetables) using low volume or pressure or electrodynamic spraying techniques, or any other treatment method known in the art or industry.
In certain embodiments, whether or not agricultural or related to food processing, compositions and/or articles or products impregnated with and/or incorporating compositions of this invention may be in the form of dustable powders or granules comprising a solid diluent or carrier, for example, fillers (also such as animal or cat litter), kaolin, bentonite, kieselguhr, dolomite, calcium carbonate, talc, powdered magnesia, fuller's earth, gypsum, diatomaceous earth, china clay and other impregnatable materials. Such granules can be preformed granules suitable for application without further treatment. These granules can be made either by impregnating pellets of filler with an inventive composition or another active ingredient or by pelleting a mixture of the active ingredient and powdered filler. For instance, compositions for dressing seed may include an agent (for example, a mineral oil) for assisting the adhesion of the composition to the seed; alternatively the active ingredient can be formulated for seed dressing purposes using an organic solvent. The compositions may also be in the form of wettable powders or water dispersible granules comprising wetting or dispersing agents to facilitate the dispersion in liquids. The powders and granules may also contain fillers and suspending agents. Alternatively, the compositions may be used in a micro-encapsulated form. They may also be formulated in biodegradable polymeric formulations to obtain a slow, controlled release of the active substance.
Regardless, such solid formulations comprising such an inventive composition can be provided in a range of products or articles in varying forms, shapes or moldings, including but not limited to cylinders, rods, blocks, capsules, tablets, pills, pellets (e.g., also pet foods), strips, spikes and the like. Alternatively, granulated or powdered material can be pressed into tablets or used to fill a range of capsules or shells. As discussed above, any such composition of this invention, whether formulated or unformulated, can be used alone, applied to a substrate or incorporated in a product or article of manufacture for a wide range of end-use applications, including but not limited to pharmaceutical, personal, industrial and agricultural compositions and related methods of use.
Generally, a useful solid carrier component can comprise any material that is at least somewhat porous and/or can hold an aforementioned antimicrobial composition without undue swelling. Together with materials of the sort described above and elsewhere herein, examples of such carrier components include silica gels, zeolites, calcium silicate, clays, activated charcoal, alumina, allophane, vermiculite, various absorbant and/or slow release polymers, and combinations thereof, as would be understood in the art by those made aware of this invention. In certain embodiments, such a carrier component can comprise one or more clay materials—examples of which are useful in the context of this invention include, but are not limited to, attapulgite, montmorillonite, bentonite, hectorite, sericite and kaolin clays and mixtures thereof. Without limitation, bentonite clays, such as those comprising colloidal hydrated aluminum silicate containing varying quantities of iron, alkali and/or alkaline earth metals, have been found especially useful. Bentonite clay materials and related processed products are commercially available from a number of sources, including American Colloid Company of Arlington Heights, Ill., under the trade name Bentonite AE H, together with other sources identified herein or as would be known to those skilled in the art.
Depending upon a particular article or end-use application, a volatile antimicrobial composition can be used neat or in combination with one or more solvents or diluent components, such as but not limited to water, aqueous alcohol and other solvents compatible with such an antimicrobial composition and/or an FFC component thereof. As a further consideration, the release or volatility of such an antimicrobial composition can be varied or adjusted by the presence of any one such solvent or diluent component.
Production of various articles of this invention generally involves admixture of an antimicrobial composition of this invention and a suitable solid carrier component. Admixture can be performed using any technique known in the art. As but one consideration, mixing technique and duration should be sufficient to disperse an antimicrobial composition over or throughout a solid carrier component. The order of admixture can vary. For instance, a solid carrier component can be provided or prepared, first, followed by addition of an antimicrobial composition. Alternatively, such an antimicrobial composition and all components used to produce a carrier component can be admixed together. With regard to the latter, the components can be admixed neat or with a solvent (e.g., water and/or an alcohol), dispersant or one or more other adjuvants. With respect to one formulation technique, the components used to produce a solid carrier and the antimicrobial composition can be admixed, with the latter, optionally, as an aqueous solution. In yet another embodiment, a powder form of a suitable carrier component can be admixed with an antimicrobial composition and a suitable binder component to provide an agglomeration of particles or granules of suitable dimension. Regardless of carrier identity, formulation technique or granule size, an antimicrobial composition can be present at about 1.0-about 3.0 wt. % of such an article and associated carrier component.
An article of this invention can be arranged and presented in conjunction with a package or enclosure for release or volatilization of an antimicrobial composition. A tote, lid, insert, covered tray or cup, carton, vial and other such enclosures known in the art can be used, providing sufficient article retention and antimicrobial release/vaporization therefrom. Examples of useful gas/vapor permeable enclosures include those configured in the shape of a flexible bag, pack or pouch of a mesh or non woven fabric composed of a gas permeable material.
The articles described herein are useful in affecting microbial activity, including inhibition of microbial growth, on and/or near a food item, thereby extending the shelf life of the food item. Toward that end, such an article—whether alone, without an enclosure or optionally presented in conjunction with a flexible bag or pouch—can be positioned with respect to or dropped in a container for shipping, storage or display of a desired food item (e.g., without limitation, fruits, vegetables and other agricultural produce), such container selected or designed depending on a particular food item. Article placement within such a container can be at any one or more points along a corresponding food supply and distribution chain.
The following non-limiting examples and data illustrate various aspects and features relating to the compositions and/or methods of the present invention, including the preparation and use of antimicrobial compositions comprising various component compounds, as are described herein. In comparison with the prior art, the present compositions and methods provide results and data which are surprising, unexpected and contrary thereto. While the utility of this invention is illustrated through the use of several compositions and component compounds which can be used therewith, it will be understood by those skilled in the art that comparable results are obtainable with various other compositions and component compounds, as are commensurate with the scope of this invention.
Fungal Isolation.
Several small stems of Ananas ananassoides were taken from a plant growing in the Bolivian Amazon in March of 2007. They were collected in a savanna region adjoining the rainforest at 12° 40′07″ S and 68° 41′58″ W and were immediately transported for analysis. Several small (2-5 inch) pieces from the stems were cut and placed into 70% ethanol for 30 seconds under a laminar flow hood. A pair of sterile tweezers was used to hold the stems separately in the flame to remove excess alcohol. Then small pieces of inner tissue (beneath the bark) were excised and placed onto potato dextrose agar (PDA) with an actively growing M. albus isolate 620 on one side of the plate having a center well removed from it. Effectively, this technique can be used to select for other isolates of Muscodor (Worapong et al., 2001a&b). During an incubation period of two weeks, the Petri plates were examined periodically for any fungal growth. Once hyphae were observed, the hyphal tips were aseptically cut out of the agar and placed on fresh PDA. The isolate was found in this manner. Several Petri plates (PDA) were used to determine if the fungus produced volatile antibiotics. This procedure included removing a 1-inch section of the agar from the middle of the plate, plating a plug of the isolate on one side and allowing it to grow for several days, and then plating test organisms on the other side of the gap.
Fungal Taxonomy.
Fungus in nature is associated with A. ananassoides and is a deuteromycete belonging to mycelia sterilia. Fungal colonies whitish on all media tested when left out of direct sunlight. Fungal colonies pinkish on all media tested when put into direct sunlight. Spores or other fruiting bodies were not observed under any conditions. Hyphae (0.6-2.7 μm) commonly growing by branching, sometimes forming perfect coils (ca. 40 μm) and having cauliflower like bodies (3.5-14 μm) associated with them. Hyphae, newly developing, grow in an undulating pattern when observed under all conditions with all of the media tested. Mycelium on PDA covers the plate in 3-4 weeks and produces a fruity odor.
Holotype:
Endophytic on A. ananassoides. Collections were made in the Bolivian Amazon in the Heath River area. The holotype comes from only one A. annisoides stem, collected in the Heath River country. A living culture is deposited as Muscodor crispans in the living Montana State University mycological collection as acquisition number 2347 (Feb. 29, 2008). Both 18S rDNA and ITS sequences of M. crispans (B-23) have been submitted to GenBank with the assigned serial number-EU195297.
Telomorph:
The telomorph of this fungus may be found in Xylariaceae, based on the similarity of the 18S rDNA gene sequence data between M. crispans and the family Xylariaceae in the GenBank database (Bruns et al., 1991; Reynolds and Taylor 1993; Mitchell et al., 1995; Guarro et al., 1999; Taylor et al., 1999). The molecular data from the 18S rDNA gene sequences of M. crispans show a 100% homology with M. albus isolate 620.
Etymology:
The genus name, Muscodor, is taken from the Latin word which means musty. This is consistent with the quality of the odor produced by the first three isolates of the genus. The species name is crispans, from the Latin meaning “curly, wavy.” The hyphae grow in regular undulating patterns.
Scanning Electron Microscopy.
Scanning electron microscopy was performed on the isolate of example 1 after procedures described by Castillo et al. (2005). Agar pieces and host plant pieces supporting fungal growth were placed in filter paper packets then placed in 2% gluteraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2-7.4) with Triton X 100, a wetting agent, aspirated for 5 minutes and left overnight. The next day the pieces were washed six×15-minute changes in water buffer 1:1, followed by a 15-minute change in 10% ethanol, a 15-minute change in 30% ethanol, a 15-minute change in 50% ethanol, five×15-minute changes in 70% ethanol, and were then left overnight or longer in 70% ethanol. They were then rinsed six times for 15 minutes in 95% and then three 15-minute changes in 100% ethanol, followed by three 15-minute changes in acetone. The microbial material was critically point dried, gold sputtercoated, and images were recorded with an XL30 ESEM FEG in the high vacuum mode using the Everhart-Thornley detector. Hyphae were measured using Image J software available on-line.
Fungal Biology. The fungus produced a white mycelium on a water based medium. No fruiting structures or spores of any kind have been found under any lab conditions. Hyphae tend to intertwine to form coils. Other species of Muscodor also have this tendency (Worapong et al., 2001a). Newly developing hyphae tend to grow in an undulating fashion rather than the typical straight pattern and commonly intertwine to make rope like structures. This pattern of growth may prove useful as a diagnostic tool in identifying this organism in in-vivo inoculation studies. The fungus also produces cauliflower-like structures that seem to be connected to the hyphae by small strands. These bodies do not germinate under any conditions and thus appear not to be spores. This observation seems to be unique for Muscodor spp. and has not been noted as being present in any other fungal species in general.
Fungal Growth and Storage.
It was determined that the isolate did not produce spores or any other fruiting bodies when several pieces of carnation leaves were placed on top of an actively growing isolate to encourage spore production, and no such structures were observed after a week of incubation at 23 C. The fungus was also plated on several different media including Cellulose Agar (CA), Malt Agar (MA), and Corn Meal Agar (CMA) to determine if spore production would be displayed. With the exception of a slower growth rate on some of the media, no other characteristics of the fungus appeared to be different, and no fruiting bodies or spores were observed.
Several methods were used to store the isolated fungus as a pure culture, one of which was the filter paper technique. The fungus was also allowed to grow on PDA, and then it was cut into small squares which were placed into vials containing 15% glycerol and stored at −70° C. The fungus was also stored at 4° C. by a similar method, using distilled water rather than glycerol. However, the most effective method of storage was on infested sterile barley seed at −70° C.
Other, more classical features of the isolated M. crispans were also examined and compared to M. albus. Muscodor crispans produced a slow growing, dense, white colored mycelium on all media tested, unless it was placed in direct sunlight, which caused the mycelium to develop a light pink color. This contrasts to M. albus that produces a whitish mycelium on all comparable media and conditions tested (Worapong et al., 2001a). The young hyphae also grew in an undulating fashion, rather than the characteristic straight cable-like fashion as commonly observed with M. albus (Strobel et al., 2001). No spores formed on any medium including ones containing the host plant material or carnation leaves. Hyphae varied in diameter (0.8-3.6 mm) and were often intertwined to make more complex structures and even hyphal coils (
Qualitative Analysis of Volatiles.
The method used to analyze the gases in the air space above a 10-day old culture of the mycelium growing in Petri plates was comparable to that used on the original isolate of the M. albus strain cz-620 (Strobel et al., 2001). First, a baked “Solid Phase Micro Extraction” syringe (Supelco) consisting of 50/30 divinylbenzene/carburen on polydimethylsiloxane on a stable flex fiber was placed through a small hole drilled in the side of the Petri plate sporting the fungal growth. The fiber was exposed to the vapor phase of the fungus for 45 min. The syringe was then inserted into the splitless injection port of a Hewlett Packard 6890 gas chromatograph containing a 30 m·0.25 mm I.D. ZB Wax capillary column with a film thickness of 0.50 mm. The column was temperature programmed as follows: 30 C for 2 min followed to 220 C at 5 C/min. The carrier gas was ultra high purity Helium (local distributor), and the initial column head pressure was 50 kPa. Prior to trapping the volatiles, the fiber was conditioned at 240 C for 20 minutes under a flow of helium gas. A 30 second injection time was used to introduce the sample fiber into the GC. The gas chromatograph was interfaced to a Hewlett Packard 5973 mass selective detector (mass spectrometer) operating at unit resolution. Data acquisition and data processing were performed on the Hewlett Packard ChemStation software system. Initial identification of compounds in the volatile mixture produced by the fungus was made through library comparison using the NIST database.
Fungal DNA Isolation and Acquiring ITS-5.8S rDNA Sequence Information.
A 10 day old culture of the present fungus, growing on PDA, was used as a source of DNA after incubation at 25° C. using the Rapid Homogenization: Plant leaf DNA Amplification Kit (Cartagen; Washington, USA). Some of the techniques used were comparable to those used to genetically characterize other M. albus isolates from Australia (Ezra et al., 2004). Squares of the cultured mycelia (0.5 cm2) were cut from one week old cultures. The agar was scraped from the bottom of the pieces, in order to exclude as much agar as possible. The pieces were placed into 1.5 ml Eppendorf vials and incubated for about 10 minutes at −80° C. The DNA was then extracted according to the instructions of the kit manufacturer. Extracted DNA was diluted (1:9) in double-distilled, sterile water and 1 μl samples were used for PCR amplification. The ITS1, 5.8S ITS2 rDNA sequence was amplified by the polymerase chain reaction using known, commercially available primers ITS1 and ITS4. The PCR procedure was carried out in a 14 μl reaction mix containing 1 μl DNA extracted from the fungal culture (1:9 dilution), 0.5 μl primer ITS1 and 0.5 μl primer ITS4, 7 μl RedMix™ plus PCR mix with 1.5 mM MgCl2(GeneChoice, Inc., Maryland, USA) and 5 μl ddH2O PCR grade (Fisher Scientific, Wembley, Western Australia, Australia). The PCR amplification was performed in a Biometra personal cycler (Goettingen, Germany): 96° C. for 5 minutes followed by 35 cycles of 95° C. for 45 seconds, 50° C. for 45 seconds and 72° C. for 45 seconds, followed by a 72° C. cycle for 5 minutes. The PCR products were examined using gel electrophoresis, on a 1.3% agarose gel for 30 minutes at 100V with TAE buffer (GelXLUltra V-2 from Labnet International, Inc., (Woodbridge, N.J., USA) or Wealtec GES cell system, from (Wealtec Inc., Georgia, USA). Gels were soaked in a 0.5 μg ml-1 ethidium bromide solution for 5 minutes and then washed in distilled water for 5 minutes. Gel imaging was performed under UV light in a Bio-Imaging System (model 202D; DNR-Imaging Systems, Kiryat Anavim, Israel). A ˜500 bp PCR product was purified using the UltraClean PCR Clean Up DNA Purification Kit (MO BIO Laboratories, Inc., California, USA). Purified products were sent for direct PCR sequencing. Sequencing was performed on both strands of the PCR product using ITS1 and ITS4 primers. Sequencing was performed using DYEnamic ET terminators on a MegaBACETM1000 analysis system (Danyel Biotech Ltd., Rehovot, Israel). Sequences were submitted to the GenBank on the NCBI web site. Sequences obtained in this study were compared to the GenBank database using the BLAST software on the NCBI web site.
Molecular Biology of Muscodor crispans.
The partial sequences of 18S rDNA, ITS1, 5.8S, and ITS2 have been demonstrated to be highly conserved regions of DNA and therefore very useful in the classification of organisms (Mitchell et al., 1995). These molecularly distinguishing partial sequences of M. crispans were obtained and compared with the data in GenBank. After searching the 18S rDNA sequences, 525 bp of M. crispans were subjected to an advanced BLAST search. The results showed 100% identity with 525 bp of M. albus (AF324337). Comparative analysis of the partial ITS 1&2 and 5.8S rDNA sequences of M. crispans hit ITS 1 and 2 of M. albus (AF324336), M. roseus (AY034664), X. enteroleuca CBS 651.89 (AF163033), X. arbuscula CBS 452.63 (AF163029), and Hypoxylon fragiform (HFR246218) at 95, 95, 90, 90, and 91% homologies, respectively.
While this invention is, in part, described in conjunction with isolated novel fungi, it will be understood that variants and mutants of such fungi—as would be understood in the art—are also contemplated in the context of the present invention. The terms “variant” and “mutant” can be defined as provided in U.S. Pat. No. 6,911,338, the entirety of which is incorporated herein by reference. Accordingly, this invention can be directed to variant or mutant strains of M. crispans and corresponding compositions thereof.
Bioassay tests for M. crispans against plant pathogens. The vapor of the volatile by-product of M. crispans was tested for microbial inhibitory activity using a relatively simple test, as previously described in the literature (Strobel, et al., 2001). A strip of agar (2 cm wide) in a standard PDA Petri dish was removed and M. crispans was inoculated and allowed to grow on one side of the plate for about a week. The test fungus or bacterium was then inoculated on the other side of the Petri dish, using small plugs of agar for the fungi. The bacteria and yeasts were streaked onto the agar (1.5 cm long). The plate was then wrapped with one piece of Parafilm and incubated at 23° C. for 48 hr. The effect of M. crispans on the growth of the test organisms and determined first by verifying the presence or absence of growth where the inoculations had taken place. If growth was observed, measurements of the diameter in two locations of the fungal hyphae were taken. The biological activity of the vapor on bacteria and yeasts were assessed by estimating the degree to which their growth was affected as percentage of growth on a control plate (Strobel et al., 2001). If no growth was observed, the test organism was aseptically removed from the test plate and inoculated onto a fresh PDA plate at some time point after exposure to the vapor in order to ascertain viability of the test organism.
Utilizing the preceding methodology, when M. crispans was grown for 7-10 days at 23° C. on PDA, the volatile by-product of the fungus proved to be lethal to several fungi and bacteria. Gram-negative and Gram-positive bacteria, as well as yeasts and each of the major classes of fungi, were utilized as test organisms. Most of the test organisms were 100% inhibited and died after a 2 day exposure to the by-product of M. crispans. (See Table 8.) Some of the test organisms did not succumb to the volatiles of M. crispans after a two day exposure, but their growth was significantly inhibited by the volatile by-product, and they were killed after a four day exposure. Such organisms include Penicillium roquefortii, Bipolaris sorokiniana, Stagonospora sp., and Fusarium oxysporum, among others.
Alternaria helianthi
Aspergillus fumigatus
Bacillus subtilis*
Bipolaris sorokiniana
Botrytis cinerea
Candida albicans*
Cephalosporium gramineum
Ceratocystis ulmi
Cochiolobolus carbonum
Colletotrichum lagenarium
Curvularia lunata
Drechslera teres
Drechslera tritici-repentis
Dreschlera portulacae
Escherichia coli*
Fusarium avenaceum
Fusarium culmorum
Fusarium oxysporum
Fusarium solani
Ganoderma sp.
Geotrichum candidum
Mycosphaerella fijiensis
Penicillium roquefortii
Phytophthora cinnamomi
Phytophthora palmivora
Pythium ultimum
Rhizoctonia solani
Saccharomyces cerevisiae*
Sclerotinia sclerotiorum
Stagonospora sp.
Tapesia yallundae
Trichoderma viridae
Verticillium dahliae
Xanthomonas axonipodis p.v.
citri*
With reference to Table 8, the effect of the vapor of the volatile by-product of M. crispans on Botrytis is quite noticeable—especially on B. cinerea, the cause of gray mold of various plants. The inhibitory and killing effects are also applicable to Botrytis allii which causes gray mold neck rot of onion. Without limitation, such results suggest the present invention can be used effectively to modify the produce surface or storage atmosphere post-harvest to prevent mold and related issues. Likewise, such results support use of an FFC composition of this invention to treat onion (e.g., Vidalia onions), shallot and garlic produce to prevent or control fungal growth.
Vapor from the volatiles of M. crispans are also effective against many of the fungi causing decay and fungal growth on grain (e.g., corn, wheat, barley, rice, etc.), and this invention can be used in conjunction with various fruits and vegetables such as potatoes, beets, carrots sweet potatoes—such grains, fruits or vegetables, whether before or after harvest, in storage or shipment. Accordingly, the compositions and methods of this invention can be applied to some of the major fungi-related issues in the agriculture and food processing fields, and can be used to target organisms such as but not limited to Alternaria, Cladosporium, Aspergillus, Penicillium, Diplodia, Fusarium and Gibberella. (See, e.g., Table 8.)
Vapor from the by-product of M. crispans was effective against the Mycosphaerella fijiensis fungus. (See, Table 8.) Accordingly, the invention can be used as treatment for the fungus-associated Black Sigatoka disease of bananas and plantains.
Citrus canker disease threatens the very existence of the United States citrus industry. As shown in Table 8, vapor from the by-product of M. crispans effectively kills the canker-causing pathogen Xanthomonas axonipodis p.v. citri. Such results suggest that FFC compositions and related methods of the present invention can be used effectively to treat seeds, seedlings, orchards, equipment or apparatus (including, e.g., worker equipment and clothing) and/or harvested fruit to prevent, inhibit or control canker disease.
As a follow up to the tests and results of Example 6, bioassay tests with the vapor of the volatile by-product of M. crispans were run against various other plant and human pathogenic fungi and bacteria. (See, Table 9, below). The fungus was grown on X-plates with PDA in one quadrant and incubated for 3-5 days at room temperature prior to inoculation with one or more test organisms. Control plates were made at the same time of inoculation and grown on the same medium that was optimal for the individual test organism. The test organisms, Staphylococcus aureus 6538, Salmonella cholerasuis 10708, Escherichia coli 11229, S. aureus ATCC 43300 (MRSA), and Vibrio cholerae ATCC 14035, were grown on Trypticase Soy Agar (TSA) in the three remaining quadrants of the X plate. Three plates of each organism, with appropriate controls, were exposed to the vapor of the by-product of the fungus for approximately two, four and six days at room temperature. In order to check for the viability of the test microbe, the fungus was then physically removed, and the control and test plates were placed in an incubator at 35±1° C. for a minimum of three to four days, with the exception of the Mycobacterium spp. which were incubated for approximately one additional month. This was done in order to ascertain if the vapor of the by-product had inhibited or killed the test organism, and viability of the organism was assessed. This same protocol was followed for the Yersinia pestis and Bacillus anthracis, except that the exposure times were changed to 3 and 5 days, and Y. pestis was incubated at 28±1° C. and in 5% CO2 after exposure to the fungus. The Mycobacterium marinum ATCC 927 was grown on 7H11 agar (Difco Co) in the remaining three quadrants, using the previously stated protocol, and incubated at 33±1° C. All three replicates in the tests with each organism behaved identically.
For all Mycobacterium tuberculosis strains, also grown on 7H11, a section of agar was removed from the plate and the B-23 fungus (on PDA) was inserted. The plates were then inoculated from a broth culture. Control plates, where no fungus was present, were also inoculated. At each appointed time interval, a section of agar was removed from the plates and transferred to a separate and empty plate and placed in an incubator at 35±1° C. in order to determine the viability of the microbe. The plates were placed in a plastic bag with moistened paper towels to prevent desiccation.
Pseudomonas aeruginosa 15442 and Burkholderia thailandensis 70038 were both grown on TSA agar. They were left at room temperature for the optimal growth time for the organism and then moved to an incubator at 35±1° C. and observed. It is to be noted that all tests using human pathogens were conducted under strict and federally approved biosafety conditions. All tests on human pathogens were repeated at least twice.
S. aureus 6538
S. cholerasuis 10708
P. aeruginosa 15442
M. marinum ATCC 927
B. thailandensis 70038
S. aureus ATCC 43300
E. coli 11229
V. cholerae ATCC 14035
Y. pestis 91-3365
B. anthracis A2084
M. tuberculosis 3081
M. tuberculosis
M. tuberculosis
M. tuberculosis
As shown in Table 9, all four acid-fast bacteria (Mycobacterium tuberculosis strains) were killed after 2, 4, 7, and 14 day exposure to actively growing M. crispans (6-10 day old culture). Other bacteria which were killed after at least 2 days of exposure to M. crispans were: Staphylococcus aureus 6538, Mycobacterium marinum, Yersinia pestis, and Salmonella choleraesuis. Relatively somewhat or completely unaffected by exposure to M. crispans were the following: Pseudomonas aeruginosa, Burkholderia thailandensis, Staphylococcus aureus (MRSA), Escherichia coli, Vibrio cholera, and Bacillus anthracis. However, the growth of S. aureus (MRSA) was only a slimy film rather than any distinct colonies and thus it was affected by the VOCs of M. crispans. In addition, the B. anthracis plate had only a few colonies left on the exposure plate, but more colonies grew after removal of M. crispans and subsequent incubation. Therefore, it is suspected that M. crispans vapor of the by-product is only effective against the vegetative cells of B. anthracis, but not against the spores. One month after the last observation time (14 days), no growth was observed on any of the plates exposed to the fungus, and growth was observed on all of the control plates.
The experiments of the following examples illustrate various embodiments of the inventive compositions and the utility thereof. One representative composition, without limitation as to component amount, concentration or ratio, is provided in Table 10. In certain embodiments, an amount of isobutryic acid can be replaced with propanoic acid at or about the same level. In certain such or other embodiments, ethanol can be replaced with acetic acid and/or 2-butanone can be replaced with either acetic acid or propanoic acid. Also, various esters can be replaced with isomers or homologs (e.g., without limitation, a 3-methylbutyl ester, of propanoic acid, for a 2-methylbutyl ester thereof) of the esters listed. The results observed in the following examples were obtained with a composition of the compounds listed in Table 10. Consistent therewith, various other compositions can be used with comparable effect.
The relative ability of the FFCs to inhibit and kill test organisms was measured. Test solutions were prepared by placing compounds in vials. The test mixture (20 microliters) was placed in a presterilized microcup (4×6 mm) located in the center of a Petri plate containing PDA. When not in use, the mixture was stored at 0 C. The test organisms (as mentioned in Table 9), freshly growing and excised on 3 mm3 agar blocks (at least 3 agar blocks per test fungus), were placed 2-3 cm from the microcup and the plate wrapped with two layers of parafilm. Measurements were made on mycelial growth from the edge of the agar blocks after a given time period. However, in the case of Geotrichum candidum they were streaked and checked for new visible growth and viability by restreaking from the original area of the agar plate that had been inoculated. Appropriate controls were also set up in which no test solution was placed into the microcup. Tests on 20 ml of the FFC mixture were done at least twice with comparable results.
Viability of the test microbes was made by aseptically removing the small agar block and placing it on a PDA plate and observing growth after 1-3 days, or by re-streaking the Geotrichum candidum on a fresh PDA plate. In this manner the viability of the microbes could be assessed. The results shown in Table 11a indicate that the organisms listed below are all inhibited by the particular FFC composition and in most cases killed by the exposure to them. These include Aspergillus niger, Penicillium sp. on cheese, Cercospora beticola, Verticillum dahaliae, Pythium ultimum, Phytophthora palmivora, Mycophaeraella fijiensis, Rhizoctonia solani, Aspergillus fumigatus, Geotrichum candidum, Trichoderma viridi, Ganoderma sp., Curvularia sp., and Botrytis alli. Thus, when properly applied, an FFC composition has an ability to control these pathogenic microbes. Such results indicate many other pathogenic microbes can be either inhibited or killed by this mixture.
Aspergillus niger
Penicillium sp. on cheese
Cercospora beticola
Verticillum dahaliae
Pythium ultimum
Phytophthora palmivora
Mycophaeraella fijiensis
Rhizoctonia solani
Aspergillus fumigatus
Geotrichum candidum
Trichoderma viridi
Ganoderma sp
Curvularia sp
Botrytis alli
With reference to the data of Table 11a, the activity profile of the FFC composition utilized indicates, in several instances, different and/or enhanced antimicrobial effect, as compared to M. crispans and vapors of the volatile by-product thereof.
With reference to the preceding example and using comparable techniques and procedures, the same pathogens were treated with propanoic acid vapors. Comparative results are shown in Table 11b, below, with the data of Table 11a reproduced in columns A and B, and observed effects of propanoic acid, alone, provided in column C (% inhibition). At 20 the amount of propanoic acid is comparable to a level of propanoic acid in certain embodiments of this invention. Propanoic acid is representative of various lone compounds of the prior art known to have certain antimicrobial effect. However, as demonstrated by comparative data of Table 11b, the present compositions provide new and synergistic results over and beyond that expected independently from a lone prior art component outside the context of this invention. As shown therein, while the prior art is at best merely inhibitory, the inventive compositions eliminate (i.e., kill) many pathogens tested. Similar results are obtainable by comparison with other such lone prior art compounds/compositions.
Aspergillus niger
Penicillium sp. on
Cercospora beticola
Verticillum dahaliae
Pythium ultimum
Phytophthora palmivora
Mycophaeraella fijiensis
Rhizoctonia solani
Aspergillus fumigatus
Geotrichum candidum
Trichoderma viridi
Ganoderma sp
Curvularia sp
Botrytis alli
Four clinical drug resistant strains of M. tuberculosis isolates (5901867, 50001106, 59501228 and 3081) were exposed to an FFC composition. For each isolate, 10 μL of the culture was placed in the middle of a 7H11 agar plate and then evenly spread across the whole surface of the plate with a sterile plastic loop. Lids from 0.65 ml microcentrifuge tubes (micro caps) were cut off and autoclaved at 121° C. for 15 minutes inside an autoclavable tube with a screw-cap lid. Sterile forceps were used to remove a micro cap which was placed in the center of the inoculated plate. The control plates (one for each isolate) did not receive a micro cap. Three plates for each isolate were made and 5, 10, or 20 μL of the FFCs were placed in each of the three micro caps of the respective plates. The plates were then placed into a zip-lock sealed plastic bag with a damp paper towel and incubated at 36° C.±1° C. for approximately 28 days. After approximately 48 hours of exposure, the micro cap was removed and disposed of and the plates were returned to the incubator. The paper towels were frequently checked and re-moistened to prevent dehydration of the media. All of the control plates had growth. All of the plates that were exposed to 5 and 10 μL of volatiles had growth. Only one isolate (50001106) exposed to 20 μL of volatiles had growth. It is to be noted that each isolate of M. tuberculosis is a clinical drug resistant strain of this organism. All experiments were carried out in US Government Approved Biosafety Laboratory Conditions.
The control plates and the plates exposed to 5 and 10 μL of volatiles were plated on 4/14/08. The plates exposed to 20 μL of volatiles were plated on 4/22/08. All plates were checked multiple times. The final check was performed on 5/19/08 and those organisms that did not survive are indicated on Table 12 as “—.”
tuberculosis
The actual effects of an FFC composition of this invention on another strain of TB are shown in
Consistent with the data of the preceding example, broader aspects of this invention can be demonstrated. Viable cultures and suitable media are prepared using materials and techniques well-known to those skilled in the art. For instance, exposure to an FFC composition of this invention (e.g., by direct contact of a liquid composition or by vapors therefrom) can result in growth inhibition or death of the following coliform bacteria (gram stain and morphology): Escherichia coli (gram negative, rod), Salmonella enteritidis (gram negative, rod), Pseudomonas aeruginosa (gram negative, rod), Staphylococcus aureus (gram positive, cocci) and Listeria monocytogenes (gram positive, rod).
Likewise, such results can also be obtained and demonstrated with various other gram-negative and/or gram-positive bacteria such as but not limited to Bacillus cereus (gram positive, rod) and Clostridium botulinum (gram positive, rod).
The IC50 was calculated for some of the test organisms that were tested against an artificial composition to mimic the volatile by-product of M. crispans. (See, Table 1.) With reference to Table 12, all of the test organisms were inhibited 100% with the utilization of 15 μL of the artificial mixture, and several of them were killed with as little as 10 μL. Verticillium dahliae, Botrytis cinerea, and Aspergillus fumigatus were not killed by even the largest volume of the mixture (30 μL), but all three were 100% inhibited with 10 or 15 μL of the test mixture. The most sensitive organism was Pythium ultimum, which was killed with 10 μL and 100% inhibited with 2.5 μL thus it is the case that the IC50 values do not necessarily reflect the killing ability of the volatiles since both P. ultimum and Botrytis cinerea possess virtually the same IC50s but one is killed and the other was not (Table 13).
Pythium ultimum
Phytophthora cinnamomi
Sclerotinia sclerotiorum
Botrytis cinerea
Rhizoctonia solani
Aspergillus fumigatus
Verticillium dahliae
Phytophthora palmivora
An artificial mixture of items, which would normally be considered as garbage, was assembled into two ammo cartridge boxes. These items consisted of waste cereal items, flower parts, meat wastes, newspaper fiber, and miscellaneous other wastes. Into one box was placed a small beaker containing 0.2 ml of the aforementioned FFC composition. Into the other box was placed a beaker with no FFCs. Both boxes were incubated for 10 days at 80° F. At the end of that time the boxes were opened and examined. It was obvious that no decay had occurred in the box with the FFCs. On the other hand, the control box had completely turned to a massive amount of decay. The use of the FFC composition for garbage treatment is an opportunity to save intact garbage form decay whilst in transit to facilities around the world that ferment the garbage into energy related products such as methane.
A vial containing 10 ml of the aforementioned FFC composition was incorporated in or with and/or used to soak a piece of clear plastic Saran® wrap 10×10 inches. The plastic wrap was soaked in the FFC composition for 6 days, drip dried and then used as a wrapper over the cheese piece thoroughly inoculated with a cheese strain of Penicillum sp. In another experiment, the cheese piece was inoculated with the fungus then wrapped with regular Saran® wrap and then injected with 10 microliters of the FFCs. The appropriate controls are indicated on the illustration above with Penicillium sp. alone, treated wrapper alone, the FFCs alone and control (no treatment). The experimental cheese pieces were incubated for 1 week at room temperature and then portions of each cheese item was eat-tested by lab personnel. It is to be noted that there was no adverse effects of storage in this manner with a prostitution of the taste of the cheese when compared to a newly cut fresh piece of cheese that had been stored in the refrigerator. The totally fungal infested cheese piece was not eaten. It is obvious from
Several yams were obtained for these experiments. It was thought that the surface contaminating microbes causing eventual decay would be plentiful enough for the inoculum. Thus, two yam pieces were placed in a plastic box with the lid sealed in the presence of a small beaker containing 0.2 ml of the FFCs. The control box contained a beaker with no FFCs. The sealed boxes were then held at room temperature for 10 days and then examined. It was obvious that no surface and deeper contamination of the treated yam pieces developed, while the control yams developed multiple areas of surface blemishes and insipient decay as illustrated in
As a related end-use application, an FFC composition and/or component thereof can be applied to harvested fruit or vegetable produce to compensate for removal of any natural, waxy or protective coating thereon. For instance, harvested squash and similar produce, with cut stems, can be treated with an FFC composition (e.g., with spray application) to control/inhibit microbial growth, improve marketability and extend shelf life.
A synthetic FFC composition of this invention, in accordance with compositions of the sort described in Tables 2-7 and 10, compared favorably with the use of live M. albus for control of seedling diseases of sugar beet (Beta vulgaris L.) caused by Pythium ultimum, Rhizoctonia solani AG 2-2 and Aphanomyces cochlioides, and root-knot nematode, Meloidogyne incognita, on tomato (Lycopersicon esculentum). The synthetic composition provided control of damping-off equal to a starch-based formulation of the live fungus for all three sugar beet pathogens, and significantly reduced the number of root-knot galls on tomato roots. Rate studies with the FFC composition utilized showed that concentrations of 2 ml/cm3 and 0.75 ml/cm3 of a soil carrier/medium component provided good control of Rhizoctonia and Pythium damping-off, respectively, of sugar beet. A concentration of 5 ml/cm3 sand provided 100% mortality in 24 h for M. incognita. By comparison, using in vitro studies, this same rate of the biorational provided fewer root-knot galls than an M. albus infested ground barley formulation applied at 5 g/l of sand.
Corynebacterium michiganese causes serious tomato loss through tissue wilt and rot. An authentic culture of this bacterium was streaked on nutirnet broth agar and a small cap was placed in the middle of the plate. Into the cap was placed 20 microliters of an artificial, lab-prepared FFC composition of this invention. A control plate contained no FFC composition. The plates were incubated for 24 hr., then examined. There was no growth of the bacterium on the FFC-treated plate. (See,
With reference to the preceding and consistent with several of the foregoing examples, a range of FFC compostions of this invention can be used either prophylactically or in the treatment of active disease states, such disease including, without limitation, diseases affecting sugar beet, tomato, onion, grain, banana and plantain, and citrus crops among others.
More generally, the present compostions and methods can be directed to the treatment and enhanced viability of seeds, plants, produce and/or related food products—whether prophylactically or in the presence of fungal or bacterial microbes, regardless of lifecycle stage (e.g., zoospore, etc.), development, growth or extent of infection. Accordingly, as would be understood by those in the art, such compositions can comprise and/or be applied, irrespective of form (e.g. powder, granules, liquid, mist, suspension, vapor, pastes, gels, coatings, etc.), on the surface of or in contact with a seed, seedling or plant (e.g., roots, stems, leaves, etc.) or produce therefrom (e.g., either pre- or post-harvest).
FFC compositions and/or components thereof, either alone or as can be incorporated into various other compositions, can be employed in a variety of end-use applications in the poultry, produce and related food-processing industries. Several such non-limiting applications are provided in the following examples.
An FFC composition of this invention, in accordance with compositions of the sort described in Tables 2-7 and 10, is used to treat a range of egg products, including but not limited to whole egg, and liquid whole egg, fortified whole egg, and liquid fortified whole egg, salt whole egg, and liquid salt whole egg, sugar whole egg, and liquid sugar whole egg, and blends of such products—whether or not liquid—with sugar, syrup solids, syrups, dextrose and dextrins and/or gums and thickening agents, together with scrambled egg mixes and liquid scrambled egg mixes, reduced cholesterol egg products and liquid products and blends thereof, and related products containing less than about 10% egg solids, shell eggs and egg components including but not limited to decholesterolized egg yolk. Such terms will be understood by those skilled in the art and have standard meanings in accordance with accepted industry and regulatory usage.
Likewise, various FFC compositions of this invention, including but not limited to those utilizing propanoic acid in at least partial substitution for isobutryic acid, can be used in the preparation and/or packaging of extended shelf life (ESL) liquid egg products, including but not limited to whole egg, scrambled mixes, egg yolk and egg white liquid products.
Likewise, various FFC compositions of this invention can be used in the processing of cracked, empty egg shells. As would be understood in the art, using available techniques and processing equipment, an FFC composition and/or a component thereof—alone or as incorporated as part of another composition—can be applied (e.g., sprayed on) to empty shells before further processing, for instance into a nutraceutical product. Likewise, one or more compositions of this invention can be applied to or incorporated with or otherwise used to treat poultry carcass, meat or a related meat product, using apparatus and techniques known in the art. By extension, one skilled in the art would understand that the present invention can also be utilized with other types of animal carcass, meat, processed meat products and all other forms of animal flesh (e.g., mammals, birds, fishes, snails, clams, crustaceans, seafood and other edible species), as also illustrated in one or more of the following examples.
As an extension of the preceding example, such an FFC composition can be incorporated into such a processed nutraceutical product (e.g., herbal and spice capsules or tablets) to inhibit bacterial/fungal growth.
While the preceding examples illustrate various downstream processing applications, the present invention can be utilized more widely in the context of egg and poultry production. Without limitation, FFC compositions or related components of this invention can be introduced to any poultry or egg production facility and/or applied to any equipment or machinery associated therewith. For instance, air or surface treatment of a coop or growing/laying facility can control, reduce and/or inhibit airborne and surface-deposited contaminants and subsequent microbial growth thereon.
An FFC composition or one or more components thereof can be incorporated into a variety of other processed food products, including food products having a water activity otherwise supportive of microbial growth. For instance, such a composition or component can be incorporated into humus, peanut butter and other such spreads, dips and mixtures. Relating to the peanut growing and processing industries, compositions and related components of this invention can be applied to peanuts before and after the shells are cracked, after an initial peanut wash, to a related processed product (e.g., peanut butter) and/or on packaging equipment and packing materials.
Likewise, an FFC composition/component of this invention (e.g., one or more of or compositions of Tables 2-7 and 10, above, or variations of the sort described therein) can be used as or incorporated into a variety of skin care or treatment products, regardless of formulation (e.g., lotion, ointment, cream, etc.).
For instance, acne is commonly caused by one or more bacterial species invading skin follicles. Demonstrating further use of this invention, an aqueous formulation of a propanoic acid-substituted FFC composition of this invention was prepared and used to treat an adolescent male subject presenting age-related acne. One application every three days for three weeks significantly reduced, by visual observation, the number and intensity of acne lesions.
Demonstrating another use of this invention in the context of a consumer and/or health care product, an FFC composition of this invention was incorporated (at approximately 2% by weight) in a representative over the counter skin cream preparation. With reference to
Likewise, this invention can be utilized in conjunction with a range of oral hygiene, care and treatment products. Without limitation, the following examples demonstrate such use of a propanoic acid-substituted FFC composition of the sort described above. Alternatively, various other FFC compositions can be used, in accordance with compositions of Tables 2-7 and 10, above, or variations thereof as described elsewhere herein.
For instance, illustrating one such oral care/hygiene product, a mouthwash/rinse product was formulated utilizing about 1% of such an FFC composition. Such a product was prepared by incorporation of such an FFC composition into a commercially-available, off-the-shelf mouthwash/rinse product. FFC compositions of this invention, regardless of concentration or dose level, can also be incorporated into a tooth paste/gel or related gum, mouth, oral or dental care product.
Lichen planus (LP) is an autoimmune disease of the skin that can occur inside the mouth or on other mucous membranes. As membranes become unstable, bacteria or fungi can take up residence in these areas and cause pain, reddening, infection, bleeding and swelling of the tissues. In order to reduce the cause of extraneous involvement of bacteria in this disease, a mouth wash product was prepared containing a 1% aqueous solution of such an FFC composition. The mouth of the patient was rinsed twice to three times daily for at least 3-4 minutes and then spit out. Photos were taken before the treatments were applied and after three weeks of treatment. After 3 weeks, the results showed an almost total reduction of gum reddening, accompanied by a nearly totally reduction of mouth and gum pain as well as a return to near normal color of the gums and other mucous membrane color. The patient reported a near-total cessation of pain/bleeding and the most relief from LP, as compared to prior experience.
A 1% solution of the aforementioned FFC composition in an off-the-shelf mouth rinse was used to reduce dental plaque and treat other problems arising from bacteria associated with oral problems. Daily use, with 3-4 mouth rinses/day, for two months resulted in little or no dental plaque build-up. Gums that were initially recorded as red, swollen, and easily caused to bleed (from notes actually taken by the dentist) now appeared as normal in color and did not bleed upon probing with the “explorer” instrument.
To confirm effectiveness of such an FFC composition, mouth spittle resulting from the previous example was placed on one side of a nutrient agar plate, spittle from a non-FFC commercial mouth rinse was placed on the other side of the same plate, and non-rinse spittle was placed on another plate. The spittles were then incubated for two days. By comparison: the non-rinse spittle had a high bacterial load; the non-FFC rinse spittle had, as expected, a reduced bacterial load; but the FFC-rinse spittle had no detectable bacteria.
In another example, an oral surgeon tested an FFC composition (e.g., as 1% of a commercial rinse/wash product) prior to oral surgery. The patient placed non-treated spittle on an agar plate (nutrient agar), rinsed with the FFC-rinse solution and placed that spittle on another agar plate. After two—three days of incubation there were no bacterial colonies on the FCC-rinse treated plate, indicating use before and after oral surgery to treat or inhibit tooth or other oral infections.
Mastitis in milk cows is caused by a complex of bacteria associated with the udder. In accordance with various non-limiting embodiments of this invention an FFC composition or a rhamnolipid modified FFC composition of the sort described below can be applied to the udder at the time of milking to reduce the prospect of bacterial infections and contamination of milk product.
Various FFC compositions of this invention can be used to reduce microbial loads on industrial/medically important biofilms. With regard to the latter, items ranging from dental prostheses to artificial joints, can be treated with an FFC composition of this invention before surgical implantation.
FFC compositions of this invention can be used to control fungal and bacterial decay of clothing items especially those exposed to moist environments (i.e., leathers, shoes, boots, straps, ties, belts). For instance, application of 0.2 ml of a 1% FFC composition of the sort described above was placed in boots that had become totally wet. The boots were enclosed to maintain the resulting vapors for a few hours, then exposed to dry air. The results showed no decay, and the boots dried without a residual moldy smell.
Compositions of the present invention can comprise various FFC components and can be formulated as would be understood by those skilled in art made aware of this invention. Without limitation, regardless of end-use application or treatment, one or more of the present FFC components and/or related compositions can be incorporated into various antibacterial or antimycotic compositions. Without limitation, such a composition can comprise a rhamnolipid surfactant component—either alone or in conjunction with an antibacterial and/or antimycotic component of the sort known in the art. With respect to the latter, such compostions can comprise a syringomycin and/or a pseudomycin component.
More specifically, as would be understood by those skilled in the art, a rhamnolipid component can comprise one or more compounds of the sort described in U.S. Pat. Nos. 5,455,232 and 5,767,090, each of which is incorporated herein by reference in its entirety. Such a rhamnolipid compound, whether presently known in the art or hereafter isolated and/or characterized, can be of a structure disclosed therein or varied, as would also be understood by those skilled in the art. For example, without limitation, whether synthetically-derived or naturally occurring (e.g., from a Pseudomonas species or a strain thereof) in an acid form and/or as a corresponding acid salt, such a compound can be alkyl- and/or acyl-substituted (e.g., methyl and/or acetyl, respectively, and higher homologs thereof) at one or more of the saccharide hydroxy positions. Likewise, whether in mono- and/or dirhamno form, any such compound can be varied by hydrophobic moiety. As a non-limiting example, with reference to
Without regard to antimicrobial or rhamnolipid identity, a carrier component of the inventive compositions can comprise a fluid selected from, but not limited to, water, an alcohol, an oil, a gas and combinations thereof. For instance, while such compositions are unlimited with respect to amount or concentration (e.g., wt. %) of antimicrobial or rhamnolipid quantities, a carrier comprising water and/or an alcohol can be used to facilitate desired formulation, shipping, storage and/or application properties, as well as effective concentration and resulting activity.
Such rhamnolipid surfactant components, antimycotic components and/or related compositions include but are not limited to those described in co-pending application Ser. No. 11/351,572, in particular examples 9-15 thereof, such application filed on Feb. 10, 2006 and incorporated herein by reference in its entirety. Such rhamnolipid surfactant components, antimycotic components and/or related compositions can incorporate or be used in conjunction with one or more FFC components and/or FFC compostions of the present invention. Such antibacterial and/or antimycotic components are known to those skilled in the art and commercially available. Various rhamnolipid components and related surfactant compositions are available from Jeneil Biosurfactant Co., LLC, under the Zonix trademark.
For instance, illustrating such rhamnolipid-related variations, a range of compositions can be prepared with one or more rhamnolipid components and one or more FFC compositions of this invention (and/or one or more FFC components thereof), for use as or in conjunction with a post-harvest wash or treatment of a wide range of fruits and vegetables. Without limitation, in such a composition, a rhamnolipid component, (e.g., as described in the aforementioned '572 application) can be present in an amount ranging from about 0.1 wt. % to about 99.9 wt. %, and an FFC composition/component (e.g., compositions of Tables 2-7 and 10, above) can be present in an amount ranging from about 99.9 wt. % to about 0.1 wt. %. With reference to applicable EPA regulations, there is no tolerance limit for the aforementioned Zonix rhamnolipid surfactants. Likewise, there is no tolerance limit for the FFC compositions/components of this invention. Accordingly, food treated with such rhamnolipid/FFC compositions can be consumed without further washing.
In accordance with the foregoing, a rhamnolipid/FFC composition can be used to wash citrus fruits. One such wash/bath composition was prepared using an 8.5% rhamnolipid solution (in water) and a 5% FFC solution (e.g., the composition of Table 10 in water). One gallon of a 95:5 (v/v) mixture was diluted to 425 gallons. Using procedural protocols known in the industry or otherwise required under applicable state or federal regulations, the composition was used effectively to clean and penetrate citrus peel—killing both surface and interior bacteria and fungi. While effective results were demonstrated with citrus fruit, this and related rhamnolipid/FFC compositions can be used comparably in conjunction with post-harvest wash or treatment of any fruit or vegetable (e.g., without limitation, blueberries, tomatoes, grapes, onions, sugar beets, sweet potatoes, apples, pears, pineapples and various other tropical produce such as but not limited to noni and acai fruit, etc.). Fruits/vegetables washed or treated with FFC compositions of this example would be recognized as safe and hygienic for human consumption.
Whether or not having an incorporated rhamnolipid component, various FFC compositions of this invention can be used to treat various fruits and vegetables (e.g., without limitation, pears, peaches, apples, tomatoes, apricots, mangos and the like) before or upon packaging or canning to reduce bacterial/fungal loads.
Sourcing of FFC Component Compounds.
Component compounds for use in compositions of this invention can be obtained commercially or prepared using synthetic techniques of the sort well-known or otherwise described in the literature. (See, e.g., U.S. Pat. No. 6,911,338, the entirety of which is incorporated herein by reference.)
Alternatively, as can be preferred in conjunction 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, GRAS component compounds and related FFC compositions of this invention can be derived naturally through fermentation techniques, and are available under the Flavorzon trademark from Jeneil Biosurfactant Co., LLC of Saukville, Wis. Accordingly, various compositions of this invention, depending on end-use or application, can comprise compounds derived from bacterial fermentation, compounds chemically synthesized and various mixtures of compounds of fermentative and synthetic origin.
With reference to the preceding, the following examples illustrate non-limiting use or incorporation of one or more compositions of this invention, such use or incorporation as would be understood by those skilled in the art made aware of this invention, and described in the context of several prior patents, each of which is incorporated herein by reference for purpose of demonstrating that one skilled in the art would understand such use or incorporation of this invention.
Illustrating other embodiments, various compositions of this invention can be formulated for use as an additive for a fruit drink, such as described in the incorporated U.S. Pat. No. 6,566,349. For instance, compositions of this invention may be added to a juice in combination with or as a substitute for a flavonoid compound and/or an antioxidant, or may be pre-applied to fruits and vegetables before processing, to increase product shelf life. As would be understood by those skilled in the art, such compositions of the '349 Patent can be modified to include one or more compositions of the present invention in an amount of which for any end-use application can be determined, in a straight-forward manner without undue experimentation.
Compositions of the present invention can also be formulated for use in preserving tea and tea/fruit mixture beverages, such as described in the incorporated U.S. Pat. No. 5,866,182. For instance, compositions of the present invention may be used in combination with or as a replacement for K-sorbate and/Na-benzoate, ascorbic acid, and dimethyl dicarbonate. As will be understood by those skilled in the art, such beverages of the '182 Patent (e.g., example 1 thereof) may be modified to include one or more compositions of the present invention, an amount of which for any particular application may be determined in a straight-forward manner without undue experimentation.
Compositions of the present invention can also be formulated for use in preserving and/or enhancing the antimicrobial effect of antiperspirants and deodorants, such as described in the incorporated U.S. Pat. No. 5,176,903. For instance, compositions of the present invention can be used in combination with or as a replacement for parabens, imidazolidinyl urea, quaternium-15, benzyl alcohol, phenoxyethanol, and various other suitable preservatives (e.g., as described in examples 1-3 thereof) and added to such antiperspirant/deodorant to protect against degradation, extend shelf life and/or enhance effectiveness, one or more such compositions in an amount of which can be determined in a straight-forward manner without undue experimentation by one having ordinary skill in the art.
Compositions of the present invention can also be formulated for use in antiperspirants, such as described in the incorporated U.S. Pat. No. 4,548,808. For instance, one or more compositions of the present invention can be added to the substantially anhydrous non-alcoholic antiperspirant products described in the '808 Patent (e.g., examples 1-6 thereof) in effective amounts readily determined without undue experimentation by one having ordinary skill in the art—to extend shelf-life and enhance antimicrobial effect.
Compositions of the present invention can also be formulated for use in animal/pet food, for example dog food, such as described in the incorporated U.S. Pat. No. 3,119,691. One having ordinary skill in the art would recognize that one or more of the present compositions can be added to low hydration dog food, high moisture dog food, and rehydratable dog food to (e.g., to the product formulations described therein) prolong the shelf-life of products disclosed in the '691 Patent, such composition(s) in an amount readily determined without undue experimentation.
Compositions of the present invention can also be formulated for use in cat litter, such as described in the incorporated U.S. Pat. Nos. 5,060,598 and 4,721,059. Various absorbent materials, including, for example, clay, alfalfa, wood chips, and saw dust, and increased absorbent materials including clay-like filler ('059 Patent) and peat ('598 Patent) are used to absorb urine and control odor. One or more compositions of the present invention may be used in conjunction with these materials (e.g., sprayed on or otherwise incorporated into) to reduce or eliminate microbial activity and control odor after use of the litters, such composition(s) in an amount readily determined without undue experimentation.
Compositions of the present invention can also be formulated for use in spray disinfectant applications, such as described in the incorporated U.S. Pat. No. 6,250,511. The '511 Patent describes including a treatment solution in the spray bottle comprising between about 25% and 75% of at least one glycol compound, between 0.2% and 60% of an antimicrobial component, between about 5% and 45% of a surfactant, and optionally effective amounts of fragrances, dyes and other additives (at col. 3 thereof). For instance, one or more compositions of the present invention can be used in conjunction with a disinfectant of the '511 Patent as a replacement for the antimicrobial component, or as an additive thereto, such composition(s) in an amount readily determined by one skilled in the art without undue experimentation.
Compositions of the present invention can also be formulated for cleaning and/or disinfecting food and beverage processing equipment, such as described in the incorporated U.S. Patent No. RE 40,050. While the '050 Reissue teaches a halogen dioxide composition, such a formulation could be modified by one skilled in the art to substitute one or more compositions of the present invention, such composition(s) in an amount readily determined without undue experimentation and contacted with or applied to such processing equipment using apparatus and techniques of the sort described in the '050 Reissue (e.g., as described in cols. 3-4 thereof).
Compositions of the present invention can also be formulated for use in preserving wood, such as described in the incorporated U.S. Pat. No. 4,988,576 (and for lignocellulosic-based composites described in incorporated U.S. Pat. No. 7,449,130). The '576 Patent teaches impregnating wood with a solution of a preservative composition comprising a graft copolymer of lignosulfonate, hydroxyl benzyl alcohol and a metal salt or a mixture of metal salts, or alternately of at least one metal salt of a graft copolymer of lignosulfonate, the copolymer being a reaction product of lignosulfonate and acrylic monomers. For instance, one or more compositions of the present invention may be used alone or in combination with such preservatives taught by the '576 Patent (or the '130 Patent), as described, respectively, in examples 1-4 and 1-2 thereof, to impregnate and preserve wood, such composition(s) in an amount readily determined by one having ordinary skill in the art without undue experimentation.
Compositions of the present invention can also be formulated for use with sanitizing and/or disinfecting wipes, such as described in the incorporated U.S. Pat. No. 4,575,891, which teaches a pad partially saturated with a disinfectant (e.g., col. 2 thereof). The '891 Patent describes suitable disinfectants as alcoholic solutions, and other antiseptic solutions. For instance, one or more compositions of the present invention may be used alone or in combination with such disinfectants and incorporated into such a wipe material, such composition(s) in an amount readily determined and incorporated by one skilled in the art without undue experimentation.
Compositions of the present invention can also be formulated for use with a hand sanitizing lotion, such as described in the incorporated U.S. Pat. No. 6,187,327. For instance, one or more compositions of the present invention can be formulated to be added to and work in conjunction with the lotion of the '327 Patent or to replace any of the active ingredients of the lotion to improve antimicrobial effect. The '327 Patent also discloses various other known hand sanitizers (e.g. an amphoteric-cationic surfactant, a cationic surfactant, a wetting agent, and a nonionic regressing agent). Regardless, a composition of the present invention can be incorporated as a replacement for or use in conjunction with any of the active ingredients in any such hand sanitizer, such composition(s) in an amount readily determined without undue experimentation.
Compositions of the present invention can also be formulated for use in treating edible or crop seeds, such as described in the incorporated U.S. Pat. No. 4,581,238, which teaches contacting with seeds with steam having a sorbate dispersed therein (e.g., in cols. 2-5 thereof). For instance, using techniques and apparatus disclosed therein, one or more compositions of the present invention can be volatilized or otherwise applied to such seeds, such composition(s) in an amount readily determined by one having ordinary skill in the art without undue experimentation.
Compositions of the present invention can also be formulated for use in preventing or inhibiting the growth of spoilage organisms, such as described in the incorporated U.S. Pat. No. 4,356,204, which teaches contacting food with an effective growth inhibiting amount of a ketohexanoic acid (e.g., in cols. 2-3 thereof). One or more compositions of the present invention can be used alone or with such a ketohexanoic acid to further inhibit and/or kill spoilage organisms. Likewise, incorporated U.S. Pat. No. 2,711,976 suggests the use of amino acids to increase the resistance of custard foods to spoilage organisms and Staphylococcus species. Again, one or more compositions of the present invention may be used alone or in combination with or as a substitute for such amino acids. Likewise, incorporated U.S. Pat. No. 2,866,819 suggests the use of sorbic acid as a preservative in foods. Again, one or more compositions of the present invention may be used alone or in combination or as a substitute for sorbic acid. Likewise, incorporated U.S. Pat. No. 2,910,368 discloses the use of EDTA with sorbic acid to increase the shelf life of vegetables. Again, one or more compositions of the present invention may be used alone or in combination with EDTA and/or sorbic acid. In each instance, such composition(s) of the present invention can be used in an amount readily determined by one skilled in the art without undue experimentation.
Compositions of the present invention can also be formulated for use in treating fruit, seeds, grains, and legumes, such as described in the incorporated U.S. Pat. No. 5,273,769, which teaches placing any of the items to be treated in a container then introducing carbon dioxide and ammonia. For instance, using apparatus and techniques described therein (e.g., examples 1-4), one or more compositions of the present invention may be utilized effectively as would be understood in the art without undue experimentation.
Compositions of the present invention may also be formulated for use in treating dental and medical articles/devices and implants, the latter as more specifically described in the incorporated U.S. Pat. No. 6,812,217, which teaches an antimicrobial polymer film applied to the exterior surface of an implantable medical device. For instance, using techniques of the sort described therein, one ore more compositions of the present invention may also be deposited on or otherwise incorporated with such a device or article (whether medical or dental) or polymer film thereon (e.g., as described in cols. 5-6) to provide antimicrobial effect, such composition(s) in an amount readily determined by one of ordinary skill in the art without undue experimentation.
Compositions of the present invention may also be formulated for use in treatment of textiles, such as in the incorporated U.S. Pat. No. 5,968,207, which teaches application of triclosan ester to textile fibers or fabric by diffusion or impregnation. For instance, one or more compositions of the present invention may be formulated for use alone or in combination with such compound to improve anti-microbial properties of a textile or fibers thereof, whether a man-made, natural, or a blend (e.g., as described in cols. 2-3 of the '207 Patent), such composition(s) in an amount readily determined by one of ordinary skill in the art without undue experimentation.
Compositions of the present invention can be formulated for treatment of surfaces of a food processing facility, related equipment and foodstuffs, such as described in the incorporated U.S. Pat. No. 7,575,744. For instance, using techniques and apparatus of the sort described therein, one or more compositions of the present invention may be formulated and disposed on equipment and foodstuff surfaces in a wide range of food processing facilities to reduce or eliminate microbial activity, such facilities/equipment including but not limited to snack, poultry, citrus, peanut and related food processing facilities/equipment (see, e.g., col. 20). Such composition(s) can be employed in an amount readily determined by one skilled in the art without undue experimentation.
Compositions of the present invention can also be formulated for use in the treatment of microbial-related diseases (i.e., mastitis, hoof & mouth, etc.) in farm animals and livestock, and to inhibit microbial growth on crops, plants, grains, and other foodstuffs, such as described in the incorporated U.S. Pat. No. 7,192,575, which teaches application of and a composition comprising clove bud oil, eucalyptus oil, lavender oil, tea tree and orange oil. For instance, one or more compositions of the present invention can be formulated for use alone or in combination with that of the '575 Patent (e.g., examples 1-2 thereof), such composition(s) in an amount readily determined by one of ordinary skill in the art without undue experimentation.
Compositions of the present invention can also be formulated for use in preserving foodstuffs such as dressings, sauces, marinades, condiments, spreads, butters, margarine, dairy based foods, and the like from microbial spoilage, such as described in the incorporated U.S. Pat. No. 6,156,362, which teaches a combination of antimicrobial components. One or more compositions of the present invention can be formulated for use alone or in combination with one or more of the components of the '362 Patent (e.g., examples 1-4 thereof), such composition(s) or in an amount readily determined by one of ordinary skill in the art without undue experimentation.
Compositions of the present invention can be formulated for incorporation with a wide range of water-based and organic-based paints, stains and related surface coatings, such as described in the incorporated U.S. Pat. No. 7,659,326 and the authorities recited therein (e.g., Kirk-Othmer-Paint; pp. 1046-1049, Vol. 17; 1996, by Arthur A. Leman, the disclosure of which is also incorporated herein by reference in its entirety). For instance, one or more compositions of the present invention may be formulated for use or alone in combination with another antimicrobial component described in the detailed description and examples 1 and 3 of the '326 Patent, such composition(s) in an amount readily determined by one skilled in the art without undue experimentation.
Compositions of the present invention can also be formulated for use or incorporation into after-shave products, such as those described in the incorporated U.S. Pat. No. 6,231,845. For instance, one or more compositions of the present invention can be used in conjunction with components of the sort described in examples 1-6 of the '845 Patent, to provide antimicrobial effect to such after-shave products of the prior art. Such compositions can be present in an amount readily determined by one skilled in the art without undue experimentation.
Compositions of the present invention can also be formulated for use or incorporation into a product for treatment of a carcass, meat or meat product (e.g., of mammals, birds, fishes, clams, crustaceans and/or other forms of seafood, and other edible species), such as described in the incorporated U.S. Pat. No. 7,507,429. For instance, one or more compositions of the present invention may be formulated for use alone or in combination with another antimicrobial component, for incorporation into a product of the sort described in the '429 patent. Such composition(s) can be present in an amount readily determined by one skilled in the art without undue experimentation, and the corresponding product(s) can be applied or otherwise utilized with techniques and apparatus described in the '429 patent or as would otherwise be understood by those skilled in the art made aware of this invention. (See, e.g., the meat processing, spraying, immersing and treating, and composition and component sections of the detailed description of the '429 Patent.)
Compositions of the present invention can also be formulated for use or incorporation into a material (e.g., a material for a coating or other incorporation) for a food product, such products including but not limited to snack foods, cereal foods and other food components, such snack and cereal foods and materials of the sort described in the incorporated U.S. Pat. No. 7,163,708. Without limitation as to how such materials can be applied, one or more compositions of the present invention can be used alone or in conjunction with one or more of the antimicrobial or preservative components of such materials, as described in the detailed description of food products and coating materials, of the '708 patent. Accordingly, as would be understood by one skilled in the art, such a composition can be present in an amount readily determined without undue experimentation.
Compositions of the present invention can be formulated for incorporation with a variety of edible spread compositions, including but not limited to peanut butter compositions, such as those described in the incorporated U.S. Pat. No. 7,498,050. For instance, as would be understood by one skilled in the art, one or more compositions of the present invention can be used in conjunction with such edible spread products to provide or otherwise enhance antimicrobial effect, as described in examples 1-2 of the '050 Patent, such composition(s) as can be present in an amount readily determined without undue experimentation.
Compositions of the present invention can be formulated for incorporation with a wide range of pest control compositions, such as those described in the incorporated U.S. Pat. No. 6,720,450 (e.g., in sections 2-3 of the detailed description thereof). For instance, one or more compositions of the present invention may be formulated for use alone or in combination with another antipesticidal component, such as that described in the '450 patent. Likewise, one or more compositions of this invention can be formulated as described therein, with a suitable carrier component, for use against various blood-imbibing insects, including but not limited to various types of mosquitoes, and insect pests of agricultural crops. The present compositions can be used as described therein for direct contact, inhibition and/or elimination of mosquitoes, including the larvae, pupa and/or adult forms thereof. Alternatively, the present compositions can be used and/or formulated for repellent action. Regardless, such composition(s) can be present in an amount readily determined by one skilled in the art without undue experimentation and can optionally include a surfactant component. Such a surfactant can be a biosurfactant. Without limitation, such a biosurfactant can be selected from monorhamnolipids, dirhamnolipids and combinations thereof.
With reference to paragraphs [0046]-[0047], [0051]-[0052] and [0105] and alternate compositions of the sort described in and available through use of the FFCs of Tables 2-7 and 10, the following examples demonstrate the use of several such FFC compositions and related articles, in accordance with this invention.
Several assays were undertaken using representative antimicrobial compositions against various test organisms. Plugs of inoculum (3×3 mm) were streaked at a minimal level, then exposed, respectively, to vapors from compositions B-L placed in a microcup center well. Radial growth from each plug was measured (mm) after 38 hours at room temperature.
Pythium
Rhizoc
Vert
Asp
Phyto
Fus
Botr
Scl
Bacillus
E. coli
Test Organisms: Pythium ultimum (Pythium); Rhizoctonia solani (Rhizoc); Verticullum dahliae (Vert); Aspergillus fumigatus (Asp); Phytophthora cinnamomi (Phyto); Fusarium solani (Fus); Botrytis cinerea (Botr); Sclerotinia sclerotiorum (Scl); Bacillus subtilus (Bacillus); and Escherichia coli (E. coli).
As demonstrated above, specific compositions of this invention can be designed for differential antimicrobial effect. For instance, while composition B was somewhat less than advantageous against the Botrytis and Sclerotinia species, composition D inhibited growth completely, under the assay conditions employed. Regardless, an antimicrobial composition of this invention—whether used neat, in vapor form or incorporated with a carrier component—can show beneficial results with a propanoic acid component present at a ratio of at least about 7:about 1 with respect to any other composition component. (See
In accordance with and illustrating use of various articles of this invention, granules of bentonite clay (e.g., from Al Harvey of Lovell, Wyo.) were impregnated with a representative composition of Table 10, above, (˜0.80 ml/g of bentonite) and placed in an open-top enclosure. Granules of this article and part of a bunch of raspberries (i.e., a post-harvest perishable food item) were placed in a sealed container. After seven days, little mold or other microbial growth was evident. (See,
Various other compositions of this invention, including without limitation compositions B-L of Example 52, can be incorporated with a solid carrier component. For instance, commercially-available bentonite clay granules can be impregnated with such a composition, for use in conjunction with a vapor-permeable enclosure, such as a sealed or re-sealable flexible bag or pouch. Whether a suitably-dimensioned mesh or of a porous non-woven Tyvek® or functionally-similar material, such a bag/pouch can provide an article with all components (i.e., an antimicrobial composition, a carrier and an enclosure) meeting FDA specifications relating to food processing and contact.
With reference to paragraphs [0055] and [0119] and Example 11, above, bentonite clay granules can be impregnated with a composition of Table 10, then packaged into a vapor permeable pouch, or similar such enclosure, and positioned proximate to a deposit or collection of human waste or refuse. Without limitation, such an article can be placed in or adjacent to a container of human waste—optionally, as a preliminary measure en route to a final treatment and/or disposal.
With reference to paragraphs [0057]-[0062] and [0119], an antimicrobial composition useful in conjunction with the end-use application of example 55 is represented, below.
Propanoic acid, 2-methyl-, methyl ester
Acetic acid, 2-methylpropyl ester
Propanoic acid, 2-methyl-, 2-methylpropyl ester
1-Propanol, 2-methyl-
1-Butanol, 3-methyl-, acetate
Propanoic acid, 2-methyl-, 2-methylbutyl ester
Acetic acid, 2-phenylethyl ester
Again, with reference to paragraphs [0057]-[0062] and [0119], another antimicrobial composition useful in conjunction with the end-use application of example 55 is represented, below.
Propanoic acid, 2-methyl-, methyl ester
Acetic acid, 2-methylpropyl ester
Propanoic acid, 2-methyl-, 2-methylpropyl ester
1-Propanol, 2-methyl-
1-Butanol, 3-methyl-, acetate
Propanoic acid, 2-methyl-, 2-methylbutyl ester
Acetic acid, 2-phenylethyl ester
Further, with reference to paragraphs [0057]-[0062] and [0119], another antimicrobial composition useful in conjunction with the end-use application of example 55 is represented, below.
Propanoic acid, 2-methyl-, methyl ester
Acetic acid, 2-methylpropyl ester
Propanoic acid, 2-methyl-, 2-methylpropyl ester
1-Propanol, 2-methyl-
1-Butanol, 3-methyl-, acetate
Propanoic acid, 2-methyl-, 2-methylbutyl ester
Acetic acid, 2-phenylethyl ester
With further reference to paragraphs [0077] and [0087]-[0091], Tables 2-7 and 10 and Examples 32-33, various FFC compositions of this invention can be used to prepare cat litter and related animal care products. For instance, bentonite clay granules or other solid carrier components are contacted or impregnated with an antimicrobial composition (e.g., 0.20 wt. % to about 10.0 wt. % or, in certain embodiments, about 1.0 wt. % to about 3.0 wt. %) of the sort represented below.
Alternatively, compositions of the sort described in Examples 52 (compositions B-L) and Examples 55a-55c can also be used in the preparation of such a litter article. Regardless, the relative amount of any such component can be adjusted for any particular formulation, desired antimicrobial effect and/or to accommodate the presence of any one or more additives of the sort described herein including but not limited to perfuming/fragrance agents.
With reference to paragraphs [0026] and [0087] and Example 52, above, compositions of this invention can be prepared using propanoic acid in conjunction with one or more acid salts, including but not limited to salts of any one or more C2-about C6 acids. Such acid salts, including those of food grade quality, can be prepared, as described below, and are available from sources well-known to those skilled in the art, including but not limited to Sigma-Aldrich (St. Louis, Mo.).
In accordance with certain embodiments of this invention and with reference to paragraph [0061], the following compositions can be considered without limitation as to component amount or concentration, such compositions including:
A. propanoic acid: a C4 acid salt:
B. propanoic acid: a C5 acid salt;
C. propanoic acid: a C6 acid salt;
D. propanoic acid: a combination of C4 acid salts;
E. propanoic acid: a combination of C5 acid salts;
F. propanoic acid: a combination of C6 acid salts; and
G. propanoic acid: a combination of C4-C6 acid salts.
Such C4-C6 acid salts and combinations thereof can be, without limitation, selected from salts of n-C4-n-C6 monocarboxylic acids and structural isomers thereof and suitable corresponding C4-C6 polycarboxylic and hydroxypolycarboxylic acids including but not limited to salts of 2-methylpropanoic acid, 2-methylbutanoic acid, 3-methylbutanoic acid, 2,3-dimethylpropanoic acid, 2,2-dimethylpropanoic acid, tartaric acid, citric acid and other C4-C6 mono- and (hydroxy)polycarboxylic acids, as would be understood by those skilled in the art made aware of this invention, such salts as can be, without limitation, selected from alkali (e.g., sodium, potassium, etc.), alkaline-earth (e.g., calcium, magnesium, etc.) and quaternary amine (e.g., ammonium, etc.) salts of such acids. Such compositions can be prepared by mixing the components neat or with a suitable solvent or diluent such as but not limited to water and/or an aqueous alcohol and, optionally, in the presence of a surface active component such as a rhamnolipid.
With reference to any composition of Example 57a, various other compositions of this invention can include one or more esters of one or more C2-about C5 acids and structural isomers thereof, in addition to or as a substitute for any such acid salt component.
With reference to any composition of Examples 57a-b, various other compositions of this invention can include one or more C2-about C8 aldehyde components in addition to or as a substitute for any such acid salt and/or acid ester component.
With reference to the compositions of Examples 57a-c, various other compositions of this invention can include another C2-about C6 acid component. Without limitation, such an additional acid component can be selected from acetic acid, isobutyric acid, citric acid and combinations thereof, in addition to or as a partial substitute for propanoic acid.
Notwithstanding the compositions of Examples 57 and 57a-d, compositions of propanoic acid and at least one C4-about C6 acid salt, compositions of propanoic acid and at least one additional C2-about C6 acid component and compositions of propanoic acid and at least one C4-about C6 acid salt providing the absence of an acid ester, aldehyde and/or ketone can be utilized. Regardless, as discussed above, any composition of this invention can be absent naphthalene and azulene derivative compounds and other fused aromatic compounds and hydro derivatives thereof.
With reference to any one or more preceding compositions of Examples 57 and 57a-e, such composition(s) can be incorporated into an article of manufacture, including but not limited to those of the sort discussed herein or otherwise used as described above. Generally, without limitation and as described above, one or more such compositions can be incorporated into a food ingredient or nutraceutical (e.g., Example 17d), a range of food products (e.g., at paragraphs [0032], [0052] and [0066]) including processed foods such as peanut butter, humus, and various dips and spreads (e.g., Example 18), cheeses (e.g., at paragraph [0067]) and other dairy and related products, solid carrier components (e.g., Examples 53-56), and such carrier components in conjunction with vapor permeable enclosures (e.g., Examples 53-55) and as a substitute for sorbic acid, benzoic acid and sorbate and benzoate salts (e.g., Examples 28 and 39).
With reference to Examples 57 and 57a-e, comparative testing of a representative composition of propanoic acid and a salt of isobutyric acid (e.g., potassium) demonstrated this invention as providing antimicrobial effect when incorporated into a food item. More specifically, 0.1 gram of potassium isobutyrate was added to 20 microliters of propanoic acid. (Similarly, several reference compositions were also prepared, as indicated in Table 15, below.) The test and reference compositions were hand-mixed into 10 grams of fresh refrigerated humus (Costco) and placed in a sealed container. The treated and control containers were held at 25° C. then examined and sampled at 10 and 18 days. Sampling of each container was accomplished with a sterile transfer needle, with approximately 1 mg. streaked onto a potato dextrose agar (PDA) Petri plate, incubated for 30 hours, then examined. Results are summarized in Table 15, below.
Purpose: Determine if a composition of this invention will inhibit mold in an enzyme modified cheese (EMC) cheddar product (EMC CH) which is susceptible to mold growth due to its high pH and low titratable acidity (TA). Procedure: Prepare a composition, obtain an EMC cheddar product, and prepare a Blue Cheese Slurry as a source of mold spores to deliver 1-10 CFU/100 gram sample. Test efficacy of the composition.
The composition of this example (designated FF #2) is comprised of potassium isobutyrate, propionic acid, acetic acid and citric acid.
It has a pH of 5.39.
1. Previous plating yielded 3.4 billion CFU/g of Penicillium roqueforti in the blue cheese.
2. Add 1 g Blue Cheese into 9 ml Sodium Citrate (2.0%) buffer ( 1/10 dilution).
3. Make one dilution ( 1/10) and 3 serial dilutions of 0.1 ml into 9.9 ml Sodium Citrate buffer.
4. Plate 0.2 ml on each sample and control “C” and “D”.
5. BM calculation to equate 6.8 CFU/sample.
Purpose: Determine if FF #2 will inhibit mold in a Lipolyzed cream product (LC) which is susceptible to mold growth due to its high pH and low TA. Procedure: Prepare FF #2, obtain Lipolyzed cream product without potassium sorbate, and prepare a Blue Cheese Slurry as a source of mold spores to deliver 1-10 CFU/100 gram sample. Test efficacy of FF #2.
Lipolyzed Cream product (LC)[Production Lot #140205]:
As shown by the results of Examples 60a-b, a representative composition of this invention can be used to effectively inhibit mold growth in otherwise susceptible dairy food products.
The composition of Examples 60a-b (FF #2) was tested against representative strains of fungi and bacteria. The composition was placed in a micro cup in the center of a potato dextrose agar plate with small plugs of inoculum spaced around the center well micro cup at 2 cm. The cup contained the amounts of the composition as indicated in Table 16. The plates were incubated for 24 hours at ambient temperature and measured. Measurements were made on a control plate and the amount of inhibition expressed for the composition is presented as the amount of growth versus control plate growth (without composition). The percent inhibition results were calculated after a minimum of two measurements for each test organism were completed.
Rhizoctonia solani
Phytophthora cinnamomi
Verticillum dahliae
Sclerotiorum sclerotiorum
Penicillum sp.
Aspergillus fumigatus
Fusarium solani
Pythium ultimum
Bortytis cinerea
E. coli
Bacillus subtilus
As a further test against the same fungi and bacteria, the composition was placed directly as a droplet at the center of a potato dextrose agar plate with small plugs of inoculum spaced around the center at 2 cm increments. The plates were incubated for 24 hours at ambient temperature and measured. The amount of composition applied is indicated in Table 17. Measurements were made on a control plate and test plates. The amount of inhibition expressed for the composition is presented as the amount of growth versus the control plate growth.
Rhizoctonia solani
Phytophthora cinnamomi
Verticillum dahliae
Sclerotiorum sclerotiorum
Penicillum sp.
Aspergillus fumigatus
Fusarium solani
Pythium ultimum
Bortytis cinerea
E. coli
Bacillus subtilus
The results of this example show that the composition is active in the gas phase, as all organisms, at one or all concentrations were affected during the course of at least 24 hours. At 25 the effect was maximized since most organisms were 100% inhibited (Table 16). When the composition was placed directly in the middle of the plate on the surface, the influence of the composition was somewhat diminished, as there was undoubtedly diffusion into the agar bed. Nevertheless, antimicrobial activity was observed (Table 17). The higher concentration (12.5 ul) affected both test bacteria and fungi more than the lower dosage (6 ul) on the agar bed, except for Verticillum.
Various compositions of the present invention were shown to have antimicrobial effect without tissue necrosis when applied to a range of fruits and vegetables sourced from local retailers and growers. With reference to the comparative tests of the following examples:
Control A: water;
Composition B was prepared with 0.2 ml propanoic acid, 0.5 g of potassium isobutyrate, 0.2 ml of isobutyl isobutyrate, and 0.2 ml of benzaldehyde (v:w:v:v) then diluted to 100 ml to provide a 1% solution (pH of about 5.5);
Composition C was prepared with 0.1 ml propanoic acid, 0.6 g of potassium isobutyrate, 0.2 ml of isobutyl isobutyrate and 0.2 ml of benzaldehyde (v:w:v:v) then diluted to 100 ml to provide 1% solution (pH of about 6.0); and
Composition D: a 1% solution of a thirteen-component composition of Table 10. For each of Compositions B and C, as well as various other compositions of this invention, isobutyl isobutyrate is a non-limiting example of a C2-05 acid ester and benzaldehyde is a non-limiting example of a C2-C8 aldehyde.
All grapes used were of the Thompson Seedless variety, and all used in the treatments were still attached to the stem. In each treatment, grape specimens were placed in a sealed plastic container box dimensioned about 6×6×2″ with a piece of Whatman no. 1 filter paper on the bottom of the box, wetted as described, below, for each. The boxes were incubated at room temperature (approx. 23 C) for one week, then assessed for decay, fungal growth and any spotting or discoloration based on the color and disposition of the skin of normal grapes.
No fungal inoculum was used in this study. Any observed degradation or decay was the result of only the normal flora present on the grapes. Digital images (not shown, but available) were taken of the grapes used in each treatment for color, size, any deformities and necrotic spots, as a basis for visual comparison.
Under the conditions used, neither Compositions B nor C resulted in any noticeable spotting or necrosis of the grape tissues.
More specifically, dipping the grapes in Composition B for a 5 minute exposure followed by incubation over a water-saturated Whatman filter paper for 1 week provided complete protection of the grapes with no decay, fungal growth, necrosis or adverse effect on the grape epidermis. (See Treatment 1). Likewise, a spray with Composition C without drying and incubation over the paper wetted with C also resulted in protection of the grapes with no necrotic spotting (see Treatment 3). (Upon completion of Treatments 1 and 3, the grapes retained texture.) However, spraying followed by drying with either C (treatment 4) or B (treatment 5) was less effective, as incubation over filter paper wetted with either C or B, respectively, resulted in some decay. The Control A fruits (treated with water), in all cases, showed decay (Treatments 2 and 6).
Based on the foregoing, both Compositions B and C can be used for protection of grapes. Dipping for 5 minutes or more followed by a rinse is an excellent protocol for disease control. Likewise, dipping or spraying the fruit and keeping it in contact with vapor of the volatile composition is also effective. There was no necrosis resulting from the treatments and no evidence of any surface spotting.
Following a procedure in accordance with the general procedure of example 62a, strawberries were placed in a sealed box for 1 week at room temperature. Beforehand, each berry of 5 was sprayed with about 0.1 ml of Control A or test Composition B or D, and the Whatman paper in the bottom of each plastic container was dampened with 0.5 ml of each composition, respectively.
Composition B, as described above, does not cause any acid-induced necrotic spots on the fruit. It also serves as a protectant to the fruit from decay-causing organisms as evidenced in digital images (not shown, but available). No decay or fungal growth was observed; there was no growth on the filter papers. Conversely, after 1 week of incubation, fruit treated with Control A rapidly showed decay, whereas with Composition D there was some decay. (The decay organisms are those intrinsic to each fruit.) It is suspected that the relatively low pH of Composition D causes some tissue breakdown and necrosis which may lead to subsequent fungal infection.
Following a procedure in accordance with the general protocol of example 62a, treated pears were placed in a sealed container for 10 days at room temperature.
After the 10-day incubation period, the pears were examined for disease symptoms as well as necrotic spotting: no necrotic flecks or spotting resulted from treatment with either Composition B or C. No disease symptoms developed in either the control or the treated pears. (Digital images are available, but not shown.)
Following a procedure in accordance with the general procedure of example 62a, cherries were placed in sealed boxes for 1 week at room temperature. (There was no fungal inoculum used in the experiment, only natural flora naturally occurring on the cherry.) Each of five cherries in each box was sprayed with about 0.1 ml of Control A or one of Compositions B and C, and the Whatman papers in the bottom of the plastic containers were dampened with 0.5 ml of each respective control or composition.
Neither Composition B nor C caused any acid induced necrotic spots or lesions one week and more after spraying. Also, each of Compositions B and C served as a protectant to the fruit: no fungal growth was observed on the fruit or on the filter paper holding the fruit. In contrast, treatment with water (Control A) resulted in rapid decay and almost total decay within a few days. (Digital images are available, but not shown.)
Whereas the fruit of this example was sprayed with one of the Compositions B and C, this and other fruits and vegetables can also be dipped/rinsed in a composition of this invention—whether at an end-point retail outlet or at any point from harvest, during storage and/or through distribution and sale, regardless of mode of application.
Following a procedure in accordance with the general procedure of example 62a, raspberries were placed in sealed boxes for 1 week at room temperature. (There was no fungal inoculum used in the experiment, only natural flora naturally occurring on the raspberries.) Each raspberry in each box was sprayed with about 0.05 ml of Control A or one of Compositions B and C, and the Whatman papers in the bottom of the plastic containers were dampened with each respective control or composition.
Again, Compositions B and C do not cause acid induced spots or lesions one week and more after spraying. Also, each of B and C served as a protectant to the fruit (i.e., no decay or fungal growth). Further, no fungal growth was observed on the filter paper holding the fruit. In contrast, treatment with Control A resulted in rapid and almost total decay within a few days. (Again, contrasting digital images are available, but not shown.)
Following a procedure in accordance with the general procedure of example 62a, black raspberries were placed in a sealed box for 1 week at room temperature. Again, no fungal inoculum was used; this study involved only flora naturally present on the berries.
The berries were placed in a plastic bag in the presence of Composition B and kept there for 5 minutes, to simulate a rinse or a soaking activity, then were placed on Whatman paper in the bottom of a plastic container. Another bunch of berries was treated likewise with water (Control A) for 5 minutes. Each plastic container was sealed, with the respective bunch of berries incubated for 1 week at 23 C.
Composition B, as described above, did not cause necrotic spots up to one week after treatment. Only traces of fungal growth were observed on one or two berries, with none on the filter paper. In marked contrast, berries treated with Control A (water) showed rapid and massive fungal decay over the incubation period. (Digital images are available, but not shown.)
Notwithstanding the treatment of this example, the compositions of this invention can be used as a dip, a spray, or as may be suitable for a particular application. For instance, such compositions can be used as a wipe or spray over cheese wheels or on smaller portions thereof for storage, sale and/or distribution.
Following a procedure in accordance with the general procedure of example 62a, cucumbers were placed in a sealed box for 7 days at room temperature. (No fungal inoculum used in this study—only flora naturally present on the cucumbers.)
Each of three cucumbers was sprayed with about 0.5 ml of either Composition B or Control A, and the Whatman papers on the bottom of each plastic container were dampened with 0.5 ml of each of A or B, respectively. After 7 days, both the control cucumbers and those treated with Composition B showed some signs of decay.
To improve application and enhance antimicrobial effect, another group of three cucumbers was soaked in Composition B for 5 minutes, and another group of three cucumbers was similarly treated with Control A. Whatman papers were prepared, for each group, as before. Each plastic container was sealed and incubated for 7 days at 23 C.
Composition B did not induce necrotic spots on the fruit for up to 7 days and more after treatment. Further, B served as a protectant to the fruit: none showed signs of decay or incipient rot. There was no fungal growth on any fruit in the group treated with B. Likewise, there was no fungal growth on the filter paper holding the fruit. In contrast, after 7 days, one of the control fruits was totally decayed, and the other two showed signs of initial decay. (Digital images are available, but not shown.)
Following a procedure in accordance with the general procedure of example 62a, mangoes were placed in a sealed box for 10 days at room temperature. (Again, no fungal inoculum was used.)
Each mango in one group of three was sprayed with about 0.5 ml of Composition B, and the Whatman papers in the bottom of the plastic container were dampened with 0.5 ml of B. Another group of three (and Whatman papers) were likewise treated with Composition C. Another group of three (and Whatman papers) was treated in the same manner with Control A (water). Each plastic container was sealed and incubated for 10 days at 23 C.
Neither Composition B nor C caused any acid-induced spots on the fruit up to 10 days and more after spraying. Also, both B and C served as a protectant, as there was no decay and no fungal growth on the respective fruit or papers holding the fruit. In contrast, one of the fruit treated with Control A was totally decayed, and initial decay was observed in the other two. (Black spots on the fruit of all three groups were due to incipient decay-causing fungi on the fruit when initially procured, and these spots began to spread in number and size during incubation of the fruit in the Control A container. There was no spreading in size or number of dark spots on the mangoes treated with either B or C. Again, digital images are available, but not shown.)
Following a procedure in accordance with the general procedure of example 62a, blueberries were placed in a sealed box for 1 week at room temperature. (No fungal inoculum was used).
A test group of blueberries was sprayed with Composition B, then placed on Whatman no 1 paper in the bottom of a plastic box. The paper absorbed the excess solution. Another group of berries was treated in the same manner with Composition C. And, a third group was treated likewise with Control A. Each plastic container was sealed, with the respective berries incubated for 10 days at 23 C.
Neither Composition B nor C caused acid-induced necrotic spots up to one week and more after spraying. However, some berries sprayed with C did have some decay, and several berries sprayed with B showed signs of decay. In contrast, the group treated with Control A exhibited almost total decay within a few days. (Digital images are available, but not shown.)
To improve application and enhance antimicrobial effect, berries from the same retail supply were used for a second study: Groups of berries were placed in a rinse of either Composition B, Composition C or Control A for 5 min then placed and sealed in boxes, as before. After 10 days, about 90% of the control fruits were colonized by fungi. Berries treated with C also had significant decay (about 50%). However, of those dipped/rinsed with B only 2-4 berries showed any decay. (As before, necrotic spots were not observed.)
Following a procedure in accordance with the general procedure of example 62a, broccoli, specifically select smaller cuttings (florets) of the floral head, was placed in a sealed box for 7 days at room temperature. (No fungal inoculum was used in this study.)
A group of florets was sprayed with about 0.5 mil of composition B, and Whatman papers on the bottom of a plastic container were dampened with B. Control A (water) was sprayed on a second grouping of florets and used to dampen underlying papers. Each container was sealed and incubated for 7 days at 23 C, with each respective grouping then evaluated for decay.
Composition B did not cause acid-induced necrotic spots on the florets 7 days and more after spraying. Also, the florets remained green throughout the test period. Also, B served as a protectant: Some slight fungal infections were noted in at least two florets, but not to the same degree as the control that had noticeable infection in almost every floret segment. In addition, the control florets faded to a light green-yellow appearance. (Digital images are available, but not shown.) Further, the florets treated with Control A showed decay around the floret base, which in many cases appeared blackened; and, in some cases, fungal hyphae were noticed on the florets. There was also some direct decay of the floret heads.
It is well known that, after picking and during storage, avocados begin to show decay at the top or stem end of the fruit. Anthracnose disease (Colletotrichum gloeosporioides) may be the cause of such symptoms. However, it is known that the fungi Dothiorella dominicana, Phomopsis spp Botryodiplodia theobromae and Lasiodiplodia theobromae can also cause avocado stem end rot. The fruit stem first becomes infected during processes involved in fruit development and ripening. A dark rot develops from the stem end as a fruit ripens after harvest. Eventually the entire fruit is subject to rot or decay. The infectious fungi are endophytic, and all are observed to have a whitish mycelium. It was thought that an antifungal treatment to inhibit and/or kill fungi in the stem end of the fruit would prevent eventual fruit decay.
Following a procedure in accordance with the general procedure of example 62a, three avocados were dipped in Control A water for 10 minutes. Another group of three avocados was dipped in composition B for 10 minutes. For each group, Whatman papers were placed on the bottom of a plastic container and dampened, respectively, with A or B. The plastic containers were sealed for 8 days at room temperature, with the fruit photographed at that time. There was no fungal inoculum used in the experiment—only flora naturally occurring on the pieces of the fruit and the stem attached to the fruit. (See preceding discussion.)
Composition B did not cause acid-induced necrotic spots on the avocado fruits 8 days after treatment. This treatment group had only minor fungal development on the stem ends, with just one fruit showing some stem end decay. There were no white mycelia on the stem ends of the treated fruit, but some slight Penicillium (green) development on one fruit. All treated fruits were firm. In contrast, a whitish mycelial decay was noted in stem ends of ⅔ of the control fruits, with massive decay in the underlying tissues of those control fruits. (Digital images are available, but not shown.)
It is to be noted that the treatment with a 10 min dip in Composition B was definitely effective in disease control. A longer dip treatment may be totally effective. Further improvements can result with use of a wetting agent (e.g., a rhamnolipid surfactant or another food grade wetting agent) in the treatment solution, whether the fruit is dipped or sprayed. (See, e.g., preceding rhamnolipid examples.) Regardless, treatment at the time of harvest will allow the solution (e.g., Composition B) to have better access to green, fresh stem ends (vs dried and shrunken), to enhance disease control.
It is well known that bell peppers, after picking and storage, begin to show decay at the top or stem end of the fruit or on bruises, cuts and scrapes of the epidermis. Once this process begins the entire fruit is subject to rot or decay. This same phenomenon occurs with other peppers, such as mini-sweet and jalapeno peppers.
Following a procedure in accordance with the general procedure of example 62a, three peppers (red, yellow and orange) were dipped in water (Control A) for 10 minutes, as a control. A second group of three was dipped in Composition B for 10 minutes. For each group, Whatman papers were placed in the bottom of a plastic container and dampened, respectively, with either A or B. The plastic containers were sealed at room temperature and photographed seven days later. (There was no fungal inoculum used in the experiment—only flora naturally occurring on the pieces of the fruit and the stem attached to the fruit.)
Composition B did not cause any acid-induced spots on the bell pepper fruits 7 days after treatment. The treatment group had only one stem end showing evidence of fungal growth, but it was minor and had not entered the fruit. All treated fruits were firm. In contrast, decay was noted in the stem end of each control pepper, with massive decay in the tissues of the orange fruit and complete collapse of the fruit. (Digital images are available, but not shown.)
Composition B may be most effective if the fruits are first rinsed with water then placed in B for 10 minutes, or longer for improved results. This might be done immediately after harvest so that the healthy tissues are still accessible to B, as the stem end of each fruit will not have had time to dry up and protect the spores and hyphae of pathogenic fungi buried in dried inaccessible tissues. If the tissue is fresh, B may have better access to these living tissues. In addition, a food grade surfactant/wetting agent may further help the access of B to tissue sites housing fungi. Furthermore, treating newly harvested fruit will also have the added advantage of allowing any scrapes, scratches, bruises or wounds to have an immediate access to the protective effects of B.
Following a procedure in accordance with the general procedure of example 62a, a group of six mini-sweet peppers was dipped in water (Control A) for 10 minutes as a control. A second group of six was dipped in Composition B for 10 minutes. For each group, Whatman papers were placed in the bottom of a plastic container and dampened with, respectfully, either A or B. The plastic containers were sealed, kept at room temperature and examined seven days later. (There was no fungal inoculum used in the experiment—only flora naturally occurring on the pieces of the fruit and the stem attached to the fruit.)
Composition B did not cause any acid induced spots on the mini sweet pepper fruits 7 days after treatment. The treatment group had only one stem end showing some fungal growth, but it was minor and had not entered the fruit. Two fruits had some minor decay on previously wounded areas in the body of the fruit, but the decay was superficial. All treated with B were firm. In contrast, decay was noted in the top end and stem of each control fruit, with additional decay in the tissues in at least three of the fruits. One of these three had completely rotted, and was believed due to Geotrichum candida. (Digital images are available, but not shown.)
Upon opening the control fruit container, there was a strong odor of totally decayed pepper fruit: It was rancid and obnoxious. After 7 days, the treated fruit smelled fresh, without such odors.
Again, Composition B may be most effective if the fruits are first rinsed with water, then placed in B for 10 minutes or longer. This should be done immediately after harvest so that the healthy tissues are still accessible to B. as the stem end of each fruit will not have had time to dry up and protect the spores and hyphae of pathogenic fungi buried in dried inaccessible tissues. If the tissue is fresh, B may have better access to these living tissues. In addition, a food grade wetting agent/surfactant further help the access of B to the tissue sites housing fungi. Furthermore, treating newly harvested fruit will also have the added advantage of allowing any scrapes, scratches, bruises or wounds to have an immediate access to the protective effects of B.
Following a procedure in accordance with the general procedure of example 62a, a group of five jalapeno peppers was dipped in water (Control A) for 5 minutes. A second group of five was dipped in Composition B for 5 minutes. For each group, Whatman papers were placed in the bottom of a plastic container and dampened with, respectively, either A or B. The plastic containers were sealed, kept at room temperature and examined eight days later. (There was no fungal inoculum used in the experiment—only natural flora naturally occurring on the pieces of the fruit and the stem attached to the fruit.)
Composition B did not cause any acid induced spots on the jalapeno fruits 8 days after treatment. In the treatment group, two stem ends exhibited some fungal growth, but it was minor and had not entered the fruit. Two fruits had some minor decay showing on previously wounded areas in the body of the fruit, but it was superficial. All treated peppers were firm. In contrast, decay was noted in the top end and stem of each control fruit, with additional decay in the tissues of each fruit. In three cases the entire fruit rotted. The majority of the fungal infections in the control group was believed caused by Geotrichum candidum. (Digital images are available, but not shown.)
It is to be noted that spray application of Composition B was not especially effective. However, as demonstrated, a 5 min dip in B was definitely effective in disease control. Longer treatment times may be totally effective.
Composition B may be most effective if the fruits are first rinsed with water then placed in B, as the stem end of each fruit will not have had time to dry up and protect the spores and hyphae of pathogenic fungi buried in dried inaccessible tissues. If the tissue is fresh, B may have better access to these living tissues. In addition, a food grade wetting agent/surfactant may further help the access of B to tissue sites housing fungi. Furthermore, treating newly harvested fruit will also have the added advantage of allowing any scrapes, scratches, bruises or wounds to have an immediate access to the protective effects of B.
Following a procedure in accordance with the general procedure of example 62a, sweet potatoes (2) were dipped in water (Control A) for 15 minutes. Another group of two potatoes was dipped in Composition B also for 15 minutes, as a rinse. Each group was placed on Whatman papers, dampened with either A or B, in the bottom of a plastic container. The plastic containers were sealed at room temperature for 7 days. (There was no fungal inoculum used in the experiment—only flora naturally occurring on the pieces of the fruit and the stem attached to the fruit.)
Composition B did not induce necrotic spots on the sweet potatoes 7 days after treatment. However, decaying fungi developed in both the treated and control potatoes. (Digital images area available, but not shown.) It is believed that the fungi had earlier infected and penetrated the hard potato exterior tissue and was not affected by surface treatment with B.
In a slight departure from the preceding, two mature eggplant fruits were sprayed (calyx end) with Composition B and incubated for 1 week at room temperature. The control fruits (2) were sprayed with water (Control A), sealed and likewise incubated. After this period, there were appreciable differences between the treated and control fruits, with the treated fruits having much less fungal growth on the calyx end of each. (Again, digital images are available, but not shown.)
Following a procedure in accordance with the general procedure of example 62a, each mid-size tomato in a group of six was sprayed with about 0.25 ml of Composition B. The control fruits (6) were sprayed with water (Control A). Each group was placed in a sealed plastic box over filter paper, dampened with either A or B, for 10 days at room temperature.
Composition B did not cause any acid-induced necrotic spots on the tomato fruits and provided complete protection from fungal and bacterial decay. In contrast, severe rot developed on the control fruits. (Digital images are available, but not shown.)
It would appear that Composition B could be applied at the time of harvest, when the fruit is packed or upon arriving at a shipping destination, and should be considered as an important tool for disease protection.
Following a procedure in accordance with the general procedure of example 62a, each large tomato in a group of five was sprayed with about 0.25 ml of Composition B. Another group of five large fruits were sprayed with water (Control A). Each group was, respectively, placed in a sealed plastic box over filter paper, dampened with either A or B, for 7 days at room temperature. (There was no fungal inoculum used in the experiment—only natural flora naturally occurring on the fruits.)
Composition B did not cause any acid-induced necrotic spots on the tomato fruits, and only one fruit showed surface tissue decay. In contrast, severe fungal growth and rot developed on each of the control fruits.
It is believed that with larger fruit, the naturally-occurring, spoiling fungi were afforded more time for penetration and infection of surface and calyx tissues, rendering subsequent treatment less effective. Earlier application, on smaller fruits, and/or use of a surfactant (e.g., a rhamnolipid) can enhance antimicrobial effect.
Following a procedure in accordance with the general procedure of example 62a, a group of cauliflower head cuttings (florets) was sprayed with about 0.5 ml of Composition B, and Whatman papers in the bottom of a plastic container were dampened with B. A water control (Control A) was sprayed onto a grouping of cauliflower florets and used to dampen filter papers at the bottom of another plastic container. After 10 days sealed at room temperature, each group of florets was evaluated for decay. (There was no fungal or bacterial inoculum used in the experiment—only flora naturally occurring on the pieces of the floral head.)
Composition B did not cause any acid-induced spots on the cauliflowers, at 10 days after spraying. Also, the florets remained strikingly white throughout the test period, and there was only a small amount of water spotting of the florets. The treated cauliflower florets remained intact and crisp. In contrast, the control cauliflower was decayed throughout and appeared as water-soaked, with a soft rot throughout the inflorescence. There was also strong yellowish discoloration on the surface. Interestingly, the entire cauliflower lost its integrity, as mere finger pressure resulted in the complete collapse of the tissues. The smell of the control container was absolutely unbearable. (Digital images are available, but not shown.)
While Composition B was applied as a spray, it can be introduced in other ways, such as by soaking shortly after harvesting. This application may be easier and less expensive, and perhaps more thorough as B may more effectively penetrate the tissues.
Following a procedure in accordance with the general procedure of example 62a, brussel sprouts (8) were dipped in water (Control A) for 5 minutes. Likewise, sprouts (7) were dipped in Composition B for 5 minutes. In both cases the fruits were placed in a sealed box for 7 days at room temperature. Whatman papers in the bottom of each plastic container were dampened with either A or B. (There was no fungal inoculum used in the experiment—only microbial flora naturally occurring on the sprouts were relied upon as infectious agents.)
Composition B did not induce necrotic spots on the brussel sprouts 7 days after treatment. The treatment group had no apparent rotted or infected sprouts. The treated sprouts appeared quite like they did a week before the time of treatment. In contrast, decay was noted in the tops ends (stems) of each control fruit, with additional decay in some of the tissues in many of the sprouts. In one case there was complete decay of the entire sprout.
The sprouts were also spray-treated, but with results not as successful as the dip treatment discussed above. (Digital images are available, but not shown.)
This sprout study illustrates that with certain fruits and vegetables a brief soak in Composition B can be more effective that a simple spray treatment. However, alternate applications can be considered. Nonetheless, wetting agents may also prove useful in some cases.
Following a procedure in accordance with the general procedure of example 62a, each of a group of bing cherries was sprayed with about 0.1 ml of Composition B, and Whatman papers in the bottom of a plastic container were dampened with 0.5 ml of B. As a control, each of another group of bing cherries was sprayed with water (Control A). Each plastic container was sealed and incubated for 1 week at 23 C. (No fungal inoculum was used—only the natural flora of the cherries were relied upon as a source of infectious agents.)
Composition B did not induce necrotic spots on the fruit up to one week and more after spraying. Also, B served as a protectant to the fruit, and no decay was observed. In contrast, the control fruit were almost totally decayed within a few days. (Digital images are available, but not shown.)
Given the positive results of previous examples, this invention was applied to a vegetable crop of a plant family not previously tested—leguminosae, represented by regular garden green beans. Following a procedure in accordance with the general procedure of example 62a, about 20 beans were treated by spray with a few mls of Composition B. Control beans (also about 20) were sprayed with deionized water (Control A). Each group was placed in a sealed plastic box over filter paper for 7 days at room temperature. Whatman papers in the bottom of the plastic containers were dampened, respectively, with either A or B. (There was no fungal inoculum used in the experiment—only natural flora naturally occurring on the beans.)
Composition B did not cause any acid-induced necrotic spots on the beans. Of the treated fruit, about 10 beans were infected, but it was almost strictly confined to the stem end of the bean. In contrast, severe rot was found wildly growing on the control beans. Almost every bean had developed some fungal infection, with infection points located anywhere on the bean. (Digital images are available, but not shown.)
In conclusion, Composition B is not toxic to the epidermis of the green bean fruits. It also appears that B, when applied to fully developed and harvestable green beans, provides useful protection from decay. B, as well as other compositions of this invention, can be applied at the time of harvest, when the beans are packed or upon arrival at a shipping destination. While spray application did not entirely inhibit infection, dipping or a longer contact with B could be more effective in disease control—as a simple spray may not impact latent infections in the fruit stem ends.
It should be noted that the procedure used in this example (and other examples) represents extremely severe storage conditions, with 100% relative humidity at room temperature for an extended time of one week. Inasmuch as fruits and vegetables are typically stored at cool temperatures and low humidity, the results of this and other examples demonstrate the enhanced antimicrobial activity available through use of this invention.
Because this invention was shown to be effective in the preservation of broccoli and Brussel sprouts it was decided to use the formula on another cruciferous vegetable crop, cabbage. A select cabbage head was cut into halves. One half served as a control and the other half was treated. Following a procedure in accordance with the general procedure of example 62a, the treated half-head of cabbage was sprayed with about 0.5 ml of Composition B. Water (Control A) was sprayed on the other half head of cabbage. Whatman papers in the bottom of each container were slightly dampened with either A or B to maintain moisture level in the container. The treated and control heads were placed in separate sealed plastic containers. After 32 days, at room temperature, the heads were evaluated for decay. (A fungal inoculum was not used—only natural flora naturally-occurring on the cabbage head.)
Composition B did not induce necrotic spots on the cabbage leaves at any time during this experiment. The leaves remained green throughout the test period and there was no decay. In contrast, the control leaves developed flecks caused by a fungus. Eventually, fruiting bodies appeared from the control head. Also, a major fungal infection developed in the stem area of the control cabbage head. (Digital images are available, but not shown.)
The results of this example nicely illustrate that Composition B, representative of various other compositions of this invention, can be used as a preservative of leafy vegetables.
With reference to a preceding example, this invention was shown to preserve large cucumbers and it was decided to test smaller pickle-sized fruit. Following a procedure in accordance with the general procedure of Example 62a, each cucumber in a group of 5 was sprayed with about 0.5 ml of Composition B. Each cucumber in a group of 6 was sprayed with water (Control A). Whatman papers were placed in the bottoms of plastic containers and dampened with about 0.5 ml of A or B, respectively. Each plastic container was sealed with its respective lid and incubated for 6 days at 23 C. (There was no fungal inoculum used in the experiment—only natural flora naturally occurring on the cucumber.)
Composition B did not cause any acid-induced necrotic spots on the fruit up to 6 days and more after spraying. Likewise, none of the treated fruits showed signs of decay or incipient rot. There was no fungal growth on any of the fruits or the underlying filter papers of the treatment group. In contrast, after six days of incubation, the control cucumbers rapidly showed fungal growth and decay. (Digital images are available, but not shown.)
Following a procedure in accordance with the general procedure of Example 62a, baby bok choy leaves were removed from their clumps. One group of 4 leaves was carefully sprayed with a few milliliters of Composition B. Another group of 4 leaves were sprayed in the same manner with deionized water (Control A). Each group was separately placed in a sealed plastic box over Whatman filter paper for 7 days at room temperature. (There was no fungal inoculum used in the experiment—only natural flora naturally occurring on the leaves.) After 7 days the leaves were evaluated for decay and discoloration.
Composition B did not induce necrotic spots on the bok choy leaves. The treated leaves, with the exception of some slight yellowing on one leaf, remained normal in appearance, without discoloration at the leaf base. (Digital images are available, but not shown.) However, the control group of leaves showed discoloration, with bacterial and fungal-induced decay on each leaf, usually originating at the leaf base.
Periodically, and all too common, Romaine lettuce is contaminated with various enteric bacteria (e.g., E. coli), causing associated food-borne illness and prompting recall from the national market place. Accordingly, the present invention was accessed for antimicrobial effect on contaminants of this important crop.
Following a procedure in accordance with the general procedure of example 62a, a group of leaves were removed from a Romaine lettuce clump, and each was carefully treated by a spray with a few milliliters of Composition B. Another group of leaves was sprayed with deionized water (Control A). Each group was placed separately in a sealed plastic box over Whatman filter paper for 7 days at refrigerator temperature (i.e., 39-40° F.), to mimic typical consumer home storage conditions. (There was no fungal or bacterial inoculum used in the experiment—only the naturally occurring flora on the leaves was relied upon to cause damage and contamination of the leaves and provide a basis for comparison.)
Composition B did not cause any acid-induced necrotic spots, and there were no bacterial or fungal-induced lesions on the Romaine lettuce leaves. Likewise, the treated leaves remained normal in appearance and were crisp after 7 days. In contrast, the control group of leaves showed discoloration with bacterial and fungal-induced decay on each of the leaves, originating at various points on the leaves. The experiment was extended to two weeks and the treated leaves continued to be crisp, non-discolored and free of infection, whereas the control leaves were rotted, discolored and not edible. (Digital images are available, but not shown.)
To further demonstrate antimicrobial effect, a small tissue sample (about 3×3 mm) was taken from discolored areas of each control leaf and plated on potato dextrose agar (PDA). As there were no such areas on the treated leaves, tissue samples were taken at random. After one day of incubation, all of the control leaf samples were observed to host either bacterial or fungal microbes or, in some cases, both. Regarding the treated leaf samples, there were no bacterial or fungal organisms evident on the PDA plate. (Again, digital images are available, but not shown.)
In conclusion, Composition B is not toxic to the epidermis of the Romaine lettuce, and when applied to harvestable Romaine leaves provides protection from bacterial/fungal decay. From the results of this example, to inhibit and/or modulate microbial activity, it would appear that a composition of this invention could be applied at the time of harvest, when the Romaine lettuce is packed or upon arrival at a wholesale or retail destination.
The most prominent post-harvest asparagus disease concern is bacterial soft rot. Decay may be initiated at the spear tip or butt end. Spears that are re-cut above the white portion of the butt end are reported to be most susceptible to bacteria. Decay can easily develop over a relatively short time, even when the harvested spears are placed in a refrigerator (crisper) box. Again, the present invention was assessed for antimicrobial activity.
Following a procedure in accordance with the general procedure of example 62a, twelve asparagus spears were lightly but thoroughly sprayed with Composition B and placed in a plastic container having a moist paper towel base. Twelve control spears were similarly sprayed with water and, likewise, placed in a plastic box. (There was no inoculum used. Only the natural flora of the spears was relied upon as a microbial inoculum.) The plastic containers were held in the crisper box of a refrigerator for varying times and evaluated on the basis of spear head integrity and odor. Evaluations were made at 7 and 10 days—times longer than the time this vegetable is typically stored prior to consumption.
Composition B did not cause acid-induced necrotic spots on asparagus spears up to 10 days after treatment. In contrast, after 1 week of incubation, seven of the control spears showed a detectable start of decay. This, of course, was accompanied by a strong odor of decay that is so common in rotting potatoes and other crops. At 10 days 8 of 12 control spears showed decay, while only 3 of the 12 treated spears showed signs of decay. (Digital images are available, but not shown.)
In summary, the modest spray of Composition B protected asparagus spears held in the crisper box (refrigerator) for up to a week. (At longer times, as reported above, decay started to manifest itself in the treated stems. The experiment was repeated with containers held at room temperature: all control spears were rotted at 5 days, but only half of the treated spears showed signs of decay.). As demonstrated, B can be used as a protectant to lengthen the shelf or storage life of asparagus. Compositions of this invention can be generally used in supermarkets as a protectant, by spray or other application, when the asparagus arrives or it can be treated in the field prior to packaging and shipment.
Following a procedure in accordance with the general procedure of example 62a, each peach in a group of six was sprayed with about 0.5 ml Composition B, and absorbent papers in the bottom of a plastic container were dampened with 1.0 ml of water. The container was then sealed. As a control, each peach in another group of six was sprayed with 0.5 ml of water (Control A), then held and sealed in the same manner. The groups of test and control peaches were incubated at 23 C for either 3 or 7 days. The experiment was done twice on two separate lots of Summer Flame peaches. It is to be noted that this test represents an extreme testing procedure because peaches are not normally held or stored under these conditions. (There was no fungal inoculum used in the experiment—only natural flora naturally occurring on the peaches being tested.)
It is observed that Composition B did not cause any acid-induced necrotic spots on the fruit over the duration of each trial. Also, B served as a protectant, as contrasted with the control which resulted in total or partial decay of all fruits in both experiments. However, in one three-day trial some decay was noted on two peaches. (Digital images are available, but now shown.)
In summary, after three days of incubation the control fruits rapidly showed decay, whereas only two of the treated peaches in one trial showed some decay. After seven days of incubation, no treated fruit showed decay whereas all fruits in the control showed signs of decay.
Regarding application of Composition B, it can be used as a rinse or drench at or close to the time of harvest followed by a drying step. This sort of treatment would eliminate the surface contaminating microbes that may eventually cause fruit decay. Another possibility is an applied spray followed by drying prior to packaging—such as in the clam shell plastic containers commonly used. Regardless, such applications, while effective, may be less than optimal against internally borne pathogens that may be present in the peach flesh—as may be a condition underlying the two treated peaches which showed some decay.
With references to Examples 63a-f, below, and more generally to various preferred compositions and applications of this invention, the term “juice” in the context of the present invention means a liquid naturally contained in fruit or vegetable tissue. Juice is usually prepared by mechanically squeezing or macerating fresh fruits or vegetables without the application of heat or solvents. Many commercial juices are filtered to remove fibre or pulp, but high pulp fresh orange juice is a popular beverage. As discussed below, juice can be in concentrate form (i.e., a concentrate), sometimes frozen, requiring additional water to reconstitute the juice. Common methods for preservation and processing of fruit juices include canning, pasteurization, freezing, evaporation and spray drying. Popular juices include, but are not limited to, apple, orange, grapefruit, pineapple, tomato, passion fruit, mango, carrot, grape, cherry, cranberry and pomegranate.
The term “nectar” in the context of the present invention means a type of non-carbonated soft drink made with fruit or vegetable juice. In some countries, the beverage industry distinguishes nectars from drinks labelled as “juice”. In the United States and the United Kingdom, the term “fruit juice” is restricted to beverages that are 100% pure juice, whereas “nectar” may contain ingredients in addition to fruit/vegetable juice. Examples of such ingredients are water (or other additional liquid), vitamins, sugar (or any kind of artificial or natural sweetener), flavors (artificial or natural), coloring agents (artificial or natural), preservatives, antimicrobial agents, thickeners, stabilizers, fibres, etc. These ingredients can be added in amounts which are common in the field of beverage making. It is preferred that all these ingredients are food grade or are allowable in food or beverage products. In addition to such ingredients, carbonated beverages can include a fruit/vegetable juice and/or nectar.
The (fruit/vegetable) juice or (fruit/vegetable) nectar formulations according to the present invention can be in a “ready-to-use” form. This means that the beverage can be consummated without any further processing (such as, for example, diluting). But it is also possible to provide juice or nectar compositions in a form which must be further processed before consumption. A very common form is a concentrate, which can be diluted either by the consumer just before consumption or by a producer, processor, distributor or retailer of an end product before packaging.
Preferred embodiments of the present invention include compositions comprising fruit or vegetable juices/concentrates and fruit or vegetable nectars. Very preferred embodiments of the present invention comprise fruit juice or fruit nectars made from oranges or grapes.
With reference to example 62a, Composition B was also used to extend the shelf life of fruit drinks. The tests of this example were conducted with commercially available orange juice. Again, no fungal or bacterial inocula were used in this experiment, only the natural flora of the juice was relied upon. Composition B was modified and reformulated with the following ratio of ingredients: 0.2 propanoic acid, 0.5 potassium isobutyrate, 0.2 isobutyl isobutyrate and 0.2 octyl acetate (v/w/v/v); the pH is 5.1, and reformulated B was tested over a range of reduced concentrations. Octyl acetate was chosen as a substitute for benzaldehyde because it constitutes the major flavor ingredient of orange juice. The juice samples were incubated at room temperature and assayed for microbial growth at 2, 3 and 4 weeks of incubation.
1. 100 ml of juice made up to 1% with Composition B
2. 100 ml of juice made up to 0.5% with Composition B
3. 100 ml of juice made up to 0.25% with Composition B
4. 100 ml of juice made up to 0.125% with Composition B
5. 100 ml of juice made up to 0.065% with Composition B
6. 100 ml of juice kept in the refrigerator at 40 C Control
7. 100 ml of juice kept at room temperature Control
At the end of each week, the solutions were examined for cloudiness and the presence of fungal colonies; i.e., fluffy inclusions in the juice. Four loops of each juice sample were streaked on half plates of PDA, then incubated at room temperature for 24 hours.
At the end of two weeks, testing all of the control and treatment cases, there were no detectable colonies of either bacteria or fungi on the streaked PDA plates. However, at three weeks the situation changed dramatically. In both the room temperature control and the 0.065% Composition B treatment the bacterial populations had increased to unacceptable levels. However, with treatments at 0.125 and 0.25% B levels there were no detectable bacterial colonies on the streaked plates. (Digital images available, but now shown.)
After 4 weeks incubation at room temperature the following was observed. The room temperature control and the 0.065% B treatment were contaminated with bacteria. The control at refrigerator temperature was free of culturable bacteria. Likewise, samples tested with 0.25, 0.5 and 1% B were free of bacteria. A small number of colonies appeared, on the PDA plate, at the 0.125% B concentration, but at an acceptable level.
As demonstrated, substitution of benzaldehyde with the octyl acetate did not significantly reduce the effectiveness of Composition B to control microbial growth in the orange juice held at room temperature for 4 weeks. Adjusting the level of octyl acetate can modify flavoring without compromising antimicrobial effect.
It is to be noted that the average stomach has a volume of about 1 quart. Drinking 1 cup of orange juice with a 0.0125% concentration of Composition B would be diluted 3-4 fold to provide an effective stomach concentration in the range of 0.03% B. At this level, based on the preceding data, it would seem that B would have little or no effect on the human gut microflora. (It should also be noted that small molecular weight organic acids are commonly found in the human gut, and the addition of such a minor amount of B will probably have no extraneous personal effect.)
With reference to the preceding example and the useful compositions demonstrated, at an effective 0.125% concentration of Composition B there are 25 microliters of octyl acetate per 100 ml of orange juice. It was found that such an amount of octyl acetate can unduly influence the orange juice taste. Taste-testing treated orange juice determined that about 5 microliters of octyl accetate was acceptable. The question is then whether B, at this level, can retain antimicrobial activity and effectively inhibit microbial contamination of the orange juice.
A room temperature test was set up with a modified Composition B at the 0.125% level containing 0.062 g of potassium isobutyrate, 0.025 ml of isobutyl isobutyrate, 0.025 ml of propanoic acid and 0.005 ml of octyl acetate per 100 ml of orange juice. (Note that this formula provides a level of octyl acetate 20 microliters less than the amount of benzylaldehyde otherwise used in B.)
The control was untreated orange juice at room temperature. Sampling was done every few days by plating 4 loops of the juice sample on plates of PDA, followed by incubation for 24 hours.
When examined for microbial contamination, there were no microbial colonies visible after 1, 2 and 3 weeks of incubation, on plates streaked either the treated or control orange juice samples. After 23 days severe fungal contamination of the control was apparent, on a streaked PDA plate. (Digital images are available, but not shown.) The control juice began to smell of fungal growth, but the treated juice remained fresh and it retained a nice orange flavor.
As demonstrated, a reduced amount of octyl acetate in Composition B is adequate to maintain antimicrobial properties and provide satisfactory taste. Testing was also done at 4 weeks and a streaked PDA plate of the treated orange juice was free of microbial contamination. In contrast, the control plate showed massive contamination of the untreated orange juice.
The study of this example was continued with sample incubation for another week, bringing the total to 5 weeks at room temperature. At the end of this time, one loop full of each of the control and treated samples was spread over the surface of 50% of a PDA plate and incubated for 1 day. The results showed that the control juice was totally contaminated with fungal growth, but only one or two colonies appeared on the treated juice plate. The treated juice had good taste and was drinkable. The control was fetid. (Digital images are available, but now shown.)
The study was continued with incubation for another week, bringing the total to 6 weeks at room temperature. At the end of this time, one loop full of each of the control and treated samples was spread over the surface of 50% of a PDA plate and incubated for 1 day. The results showed that the control juice was totally contaminated with fungal growth, but only one colony appeared on the treated juice plate. The treated juice still had a nice taste and was drinkable. The few fungal spores in the treated juice were not growing and did not produce a mycelium. (Again, digital images are available.)
The study was continued with incubation for another week, temperature bringing the total to 7 weeks at room temperature. At the end of this time, one loop full of each of the control and treated examples was spread over the surface of 50% of a PDA plate and incubated for 1 day. The result showed that the control juice was totally contaminated with fungal growth, but no colonies appeared on the treated juice plate. The treated juice still had a nice taste and was drinkable. There were no visible microbial colonies in the treated juice.
Digital images of the juice samples (not shown, but available) were recorded at 7 weeks. The room temperature control showed a large amount of contamination from fungal mycelial growth, with a prominent fungal mat on the juice surface. The treated orange juice solution had little to no microbial contamination, and regular orange juice was held in the refrigerator for the duration of the experiment. Both the treated and the refrigerated control orange juices upon shaking were perfectly tasty, and settling of pulp materials in the treated juice mimicked that of the refrigerated control.
As demonstrated, a modified Composition B using an appropriate amount of octyl acetate, as a substitute for benzaldehyde, was effective in preventing microbial growth in orange juice. The treated juice was found to be tasty and free of microbial contaminants at least up to 7 weeks at room temperature. The control juice was contaminated and unconsumable after 3-4 weeks at room temperature.
Following a procedure in accordance with the procedure of example 63b, grape juice was treated with Composition B modified to provide methyl anthranilate (MA) (a grape flavoring agent) as a substitute for benzaldehyde.
Preliminary taste testing determined that about 5 microliters of methyl anthranilate provided an acceptable taste level. Composition B was used at the 0.125% level and modified to contain 0.062 g of potassium isobutryate, 0.025 ml of isobutyl isobutyrate, 0.025 ml of propanoic acid and 0.005 ml of methyl anthranilate per 100 ml of grape juice. Note that this level of MA is 20 microliters less than the amount of benzylaldehyde used in B, a level found to be very successful in the orange juice study of the preceding example.
Both the treated and control grape juice were kept at room temperature. (No microbes were used as inocula only normal contaminating microbes were counted on as being present.) Sampling was done every week by plating 2 loops of the treated and control juice, respectively, on a half plate of PDA, followed by incubation for 24 hours.
After one week, microbial colonies were visible after plating/incubation of the control grape juice. In contrast, no colonies appeared after plating/incubation of the treated grape juice. The control juice began to smell of fungal growth, but the treated juice retained a fresh grape scent at 1 week. At two weeks, fungal contamination again appeared on the plated control juice, but not the treated juice. The actual grape juices in this experiment were photographed after two weeks of incubation. Fungal hyphae are visible in the control, but not the treated juice.
At three weeks, the juices were re-struck on PDA, and again the results were identical to what occurred at week two. There was fungal contamination of the control but not the treated juice. The same results were obtained at 4 weeks of incubation of the juice samples at room temperature, with the control contaminated and the treated juice free of microbial contamination. (Digital images are available, but not shown.)
Following a procedure in accordance with the procedure of example 63b, peach juice was treated with Composition B modified to provide gamma-decalactone (a peach flavoring agent) as a substitute for benzaldehyde.
Preliminary taste testing determined that about 5 microliters of gamma-decalactone provided acceptable taste. Composition B was used at the 0.125% level and modified to contain 0.062 g of potassium isobutyrate, 0.025 ml of isobutyl isobutyrate, 0.025 ml of propanoic acid and 0.005 ml of gamma-decalactone per 100 ml of a commercially available peach juice. Note that this level of the lactone is 20 microliters less than the amount of benzylaldehyde used in B, a level found to be very successful in the orange and grape juice, studies of the preceding examples.
Both the treated and control peach juice samples were kept held at room temperature. (No microbes were used as inocula—only normal contaminating microbes were present as the spoiling agents.) Sampling was done every week by plating 2 loops of each juice sample on a half plate of PDA, followed by incubation for 24 hours.
After one week, no microbial colonies were visible after plating/incubation of the control and the treated peach samples. However, after 2 weeks of incubation at room temperature the plated control revealed fungal contamination. There were no fungal colonies in the treated juice after plating. (Fungal colonies were also visible in the control juice, itself, while none were evident in the treated juice.) After three weeks there was massive fungal growth in the plated control, but not the treated juice. At six weeks the fungus continued to grow in the control juice, but the treated juice remained free of contamination. (Digital images are available, but not shown.)
As demonstrated, in the context of a fruit juice, the benzaldehyde component of Composition B—and, more generally, other compositions of this invention—can be substituted, whole or in part, with a flavoring agent compatible with the juice treated against microbial contamination.
With the results of example 63c, grape juice, it was thought to use a modified Composition B, containing methyl anthranilate instead of benzaldehyde, on red seedless grapes. (Again, there was no fungal inoculum used in the experiment—only natural flora naturally occurring on the grapes.)
A variation of the inventive composition used in the preceding example, 0.2 propanoic acid, 0.5 potassium isobutryate, 0.2 isobutyl isobutryate and 0.2 methyl anthranilate (v/w/v/v) was made up to 1% solution in water. Red seedless grapes were sprayed with this modified Composition B, then placed in a plastic container on Whatman paper. The control berries were sprayed with water (Control A) and, likewise, placed over Whatman paper. Each plastic container, for the control and treated grapes, was sealed and incubated for 1 week at 23 C. The containers were then incubated for 2 additional weeks at 42 F to mimic consumer refrigerator storage conditions.
Again, modified Composition B did not cause acid-induced necrotic spots on the fruit up to one week after treatment. Likewise, after 1 week, there was no fungal decay noted on any individual fruit; and there was no decay after 2 additional weeks at 42 F. In contrast, after 1 week of incubation, the control rapidly showed decay, with fungal growth apparent on nearly every grape; and the fungus grew and spread during 2 additional weeks at refrigeration temperature. As with example 62a and Compositions B and C, a modified, reformulated Composition B was shown to be effective as applied to grapes. (Again, digital images are available, but not shown.)
Given the results of the two preceding examples, with grapes and grape juice, just as modifications of Composition B were used with antimicrobial effect in orange and peach juice, corresponding compositions of this invention and variations thereof are used with comparable effect on orange and peach fruits.
Over the years, raw plant sprouts from commercial growers have been linked to several outbreaks of foodborne illness. Notable instances include:
Regarding the reported outbreaks, the likely source of the pathogen was contaminated seed. Seeds may become contaminated by bacteria in animal manure in the field or during post-harvest storage. Using animal manure to fertilize fields of alfalfa and other plants intended for non-human consumption may be hazardous if seeds from such plants are then used for sprouting. During germination, abundant nutrients, high levels of moisture, and heat generated during the sprouting process help to ensure survival and growth of bacteria external to the plant tissues.
Mishandling of sprouts during production, packing, or distribution has not been implicated as the source of sprout contamination. However, bacteria already present in the sprouting seed can continue to thrive if proper food handling techniques are not practiced during harvest, processing, and preparation. In addition for a need to control pathogenic enteric bacteria, there is a need to have safe effective agents for the control of seed-associated fungal and bacterial pathogens that have the potential to cause seedling blights, damping off and disease expression on the developing plants. Toward this end, Composition B, a representative composition of this invention, was tested for potential to control fungal and enteric bacterial pathogens associated with seeds/sprouts. Because of its size and ease in handling, the mung bean (Vigna radiata) was chosen as the seed/sprout to explore these possibilities.
The mung bean seeds were obtained from a local seed dealer. In order to determine if the seeds were carrying enteric microbes, ten seeds were placed in 10 ml of sterile water for 15 minutes. Each bean was then carefully placed on a sterile Whatman filter paper until dryness was achieved. Subsequently, each seed was placed on a Petri plate containing PDA. The incubation time was 2 days, at which time all seeds appeared free of any major bacterial contamination—an indication that this batch of seeds could be used in the enteric bacterial inoculation test described below. However, interestingly, after 5 days of incubation at least three seeds began to sport fungal growth and one seed showed bacterial growth. Thus, the seeds were not entirely free of microbial contaminants. The seed with bacterial contamination tested negative when placed on the MacConkey medium (discussed below), which is selective for enteric bacteria such as E. coli. Thus, this seed batch was deemed essentially free of enteric bacteria.
The E. coli used in this experiment was isolated from the bodily wastes of co-inventor Strobel, and was confirmed as E. coli by 16 S rDNA sequencing, to ensure that a wild type bacterium was used in the experiment, as a wild type microbe would likely be the most commonly encountered plant contaminant. (A lab type bacterium that may have undergone a loss of certain critical features upon continuous colony transfer may yield an organism that may not behave in a manner expected of E. coli.) Test results show that the 16 S sequence of the strain isolated represents a 99% match of its 16 S rDNA to that of authentic E. coli in the NIH GenBank. (Major Sequencing work was done at the School of Engineering—Biofilm Engineering Dept. at Montana State University by Heidi Smith. Other work was done by the ACGT Inc in Illinois by Dr. Hargeet Brar. The 16 S sequence is available, but not shown.)
MacConkey selective agar is a modification of Neutral Red Bile Salt Agar developed by MacConkey as a selective medium for enteric microbes. (By way of background, it was one of the earliest culture media for the cultivation, identification and isolation of enteric organisms. It has also been used in the isolation of pathogens from foods and coliforms in water samples.) The MacConkey Agar formulation presently in use is a modification of the original. In addition to containing sodium chloride, the modified formula has a lowered agar content and an adjusted concentration of bile salts and neutral red. Differentiation of enteric microorganisms is achieved by the combination of the neutral red indicator and lactose. Lactose-fermenting organisms form pink colonies surrounded by a zone of bile salt precipitation. The color change is due to the production of acid which changes the neutral red pH indicator from colorless to red. Acid production is also responsible for the formation of bile salt precipitation. Non-lactose-fermenters (Salmonella spp. and Shigella spp.) develop into transparent, colorless colonies with no precipitated zone. Peptones are incorporated into MacConkey Agar to provide amino acids and nitrogenous compounds. Sodium chloride is present to maintain osmotic equilibrium. Lactose is added as a possible carbon source for energy, and the acids produced from this activity precipitate out the bile salts. Bile salts and crystal violet are added to inhibit the growth of most gram-positive organisms.
About 300 mung bean seeds from the original batch were placed in 15 ml of distilled water containing about 2,800,000 cells of E. coli. The E. coli strain used is as described above. The seeds were then incubated for 15 min. in the bacterial suspension, placed on a towel blotter until free of moisture, then placed in a sterile positive air flow hood until dry. Half of the seeds were placed in a control water treatment consisting of 10 ml of sterile distilled water (Control A) for 15 min. The other half of the seeds were placed in 10 ml of Composition B (see Example 62) for 15 min. Each group of seeds were, respectively, placed on a damp Whatman filter paper in a sealed plastic container, then incubated for 24 hrs. at room temperature. Ten seeds were then randomly selected from the treatment group, as were ten from the control group, and they were placed aseptically onto PDA-petri plates. After 24 hrs. the seeds were examined for bacterial contamination: 8 of 10 in the control group were sporting bacterial colonies, but none in the treated group supported bacterial colonies. The observation was also made after 48 hrs. and photographed with the same result. (Each day a gentle spray of distilled water was administered to each seed group as the experiment continued.)
After 3 days of incubation on PDA, one seed in the treated group sported a Pantoea-like microbe and it was judged as a seed contaminant. This organism was negative on the MacConkey medium. Also, interestingly, after 3 days or more, 50% of the control group of germinated seeds were sporting fungal contamination, but none in the treated group showed the presence of any pathogenic fungi. This suggests that seed fungal pathogens were also controlled with this treatment—because none appeared in the Petri plates containing seeds treated with B. An examination of all of the germinated seeds, after 3-4 days, in both groups revealed that 45% of the control group possessed rootlets that were discolored. These were plated on PDA and some of the common root infecting fungi were found associated with these discolored roots, a result very close agreement with the percent of the seeds on the PDA plate that were sporting fungal pathogens. In the treated seed group, only 1-2% of the roots showed discoloration. (Digital images are available, but not shown.) As a side note, the treated mung beans germinated as well as the control group indicating that the treatment with B did not inhibit seed germination.
In the case of the control group the isolated colonies (from each of 7 of 8 seeds) met a perfect outward match for E. coli that was used as the inoculum after 3 days on PDA; that is, they were off-white or beige in color with a shiny texture. The colonies looked as if there was a mucus or a cloudy film over the whole surface of the colony. The colonies were slightly raised and had an entire, fixed margin and a steady growth pattern, creating concentric growth rings in the colony. Each of the isolates from the 7 seeds were examined by Gram staining and each revealed a Gram negative rod shaped bacterial population fitting the size and shape of standard E. coli as described in the literature as well as with the E. coli strain used to inoculate the seeds. Furthermore, when the bacterial colonies isolated from the 7 control seeds were streaked onto the MacConkey medium each grew with the classical red colony formation. The results obtained were expected for the presence of E. coli. (It is to be noted that the one bacterium isolated from the treated group of seeds was negative in the MacConkey test and only the untreated or control seeds bearing the enteric bacteria inoculation load gave strict positive reactions in the MacConkey test. (Digital images are available, but now shown.)
Although the data are conclusive for the observation that Composition B was effective in the treatment of mung bean seeds bearing E. coli infestation, additional work was done to unequivocally show that the bacterial species isolated from the control seeds in this experiment was in fact an E. coli isolate originally used as inoculum. Two of the seven isolated bacterial cultures were selected for 16 S rDNA sequencing at the MSU sequencing lab at the Biofilm center in the Engineering College. The results showed 95 and 96% identity to the 16 S rDNA to that of the E. coli starting culture. The results did suggest that the cultures isolated directly from the seeds may not have been pure, but E. coli was present. This is to be expected because the seeds with which the study was started were mostly but not entirely free of bacterial contamination, and the results are what would be expected with seeds not pretreated in any manner.
Because Composition B was effective in protecting mung bean seeds from fungal and enteric bacterial contamination, it was therefore desirable to formulate B with a suitable solid carrier that could be applied to the mung bean seed. Toward this end, bentonite clay particles were ground in a mortar with a pestle until the particles were a powder. The components of Composition B, in the ratio provided in Example 62, were added to 10 grams of bentonite powder in an amount sufficient to provide two formulations of the components of B: at a 10% level and at a 1% level. For each 20 seeds treated 1 gram of the formulated powder as control was used. Each group of seeds was placed on dampened Whatman filter paper and incubated in a sealed plastic box for three days at room temperature. At the end of two days another water spray was added. At the end of three days, 12 of 23 control seeds exhibited roots with browning and decay. There was no decay in the treated seed groups, but the group treated at the 10% level showed a retardation in seed development. Therefore, it appears the amount of composition of this invention should be controlled at a level where it is effective against fungi, but not affecting plant development.
As shown by the preceding examples, Composition B was effective as a mung bean treatment to control fungal infections of the germinating seeds, especially the newly developing rootlets. Further, B effectively eliminated the detectable presence of E. coli on mung bean seeds, demonstrating a safe, effective means of controlling enteric pathogenic bacteria that can be extended to the treatment of alfalfa, bean, and other sprouts, together with other vegetables and fruits, and seedlings, to reduce enteric bacterial contamination.
Components of Composition B can, for instance, be formulated into a solid carrier (e.g., with bentonite at the 1% level) which can be dampened with water with seed then added to it. The sticky bentonite adheres to the seed coat and releases such an inventive composition to the seed coat to protect it from infection. Other carrier or sticking agents could also be used. Almost complete control of fungal infection of the seeds was realized.
Many seeds carry the spores of plant pathogens, and germination begins the infection process. For the sprouting industry, there remains a search for a safe effective fungal control agent that will control the pathogens without harming the plant.
Toward this end, the present invention was evaluated for use in disinfecting radish seeds of fungi. Following a procedure in accordance with the procedures of Examples 62a and 64a, a set of about 100 seeds were placed in Composition B for 15 minutes. A second group of seeds was placed in water (Control A) for 15 minutes. (Soaking for a period of time was found to be more effective than spray application.) Each group of seeds was removed and placed, respectively, on a damp Whatman filter paper and incubated for 5 days at room temperature in a sealed plastic container. (A fungal inoculum was not used—only natural flora naturally occurring on the radish seeds.)
The seeds were then observed for any effects after 5 days. In the control group, 91% of the radish seedlings had a discolored, fungal infested rootlet. In contrast, in the treated group only 8% of the roots were discolored with the remainder having healthy whitish roots. Such results demonstrate the utility of this invention to modulate, inhibit or otherwise control microbial infection of plant seeds and sprouts.
Smut disease, caused by Ustilago avenae, is pervasive in all areas where oats are grown. The two smut diseases of oats—covered smut and loose smut—are very similar in appearance. When a smutted oat panicle emerges from its enclosing sheath, an olive-brown to brownish black powdery mass (sorus) of smut spores has completely replaced the oat grains, and sometimes even the awns and glumes. This smut mass is composed of many millions of spores (teliospores), contained within a delicate, whitish gray membrane. Smutted panicles do not spread as widely as normal ones. Usually all of the spikelets and heads on an infected plant are smutted. Occasionally, the panicle on the main tiller may escape or perhaps some upper spikelets in a head may appear healthy. Smutted plants are generally shorter than healthy plants and are often passed over by harvest machinery. In loose smut, the thin membrane usually breaks and disintegrates soon after the oat panicles emerge. The naked mass of smut spores is quickly scattered by wind and rain, leaving a denuded panicle.
Typically, fungicides are used to treat seed and are effective in disease control. However, with the advent of organic agriculture other alternatives will be necessary for disease control. To this end, it was decided to test a representative composition of this invention on Ustilago avenae and also gauge its effect on oat seed germination. (Digital images of results are available, but not shown.)
Healthy oat seeds and loose smut spores on diseased oats were obtained from a local producer. Composition B is as described in example 62 and was evaluated initially against an inventive composition of Table 10, designated as B-23. All tests were conducted at room temperature.
In order to determine if a basic B-23 formula had any effect on Ustialgo avenae, various amounts of it were placed into a plastic center wells of PDA plates sprayed with spores of the fungus. After several days the plates were observed: the highest amount of B-23 at 50 μl cleared the plate of all growth; at 20 μl there was severe inhibition of the fungus. However, even though it controlled the fungus, B-23 was not used, as it was thought that the acidity (low pH) would inhibit and kill the oat plants. Accordingly, studies of this example were conducted with Composition B.
Initially, 10 ml of Composition B was placed in a small plastic container and 20 mg of spores on plant material were mixed thoroughly therewith. After 15, 30, 60 and 120 minutes, bits of plant material were aseptically removed from the container with forceps and rinsed with sterile distilled water. The material was then streaked onto a plate of PDA. Correspondingly, a water control was matched with each treatment time. The plates were incubated for two days, with the results recorded.
Treatment exposure times of 15, and 30 min were less than optimal in controlling the germination and growth of Ustilago avenae. However, an exposure time of at least 1 hr fully controlled the growth of the fungus. Microscopic examination of the spores treated 1 hr revealed that none had germinated, even after having been on the plate for 2 days. By contrast, all water controls, for the designated times of 15 min., 30 min., and, 1 hr, showed growth and development of U. avenae.
To access effect on germination, oat seeds were exposed to Composition B for 1 hr, rinsed with water then plated on water agar. After two days, the germination rate was identical to that of a water control, at about 95%. Likewise, the rate of growth was identical to that of the water control; i.e. rootlet development length, root hair growth etc. These and the results of the preceding example show that, under the condition utilized, B controlled smut disease and did not cause an impact on seed development. Exposure to B for a time up to 2 hr caused some restriction in seed germination and development.
Oat seeds inoculated with spores of loose smut were treated for 24 hr in Composition B then plated on water agar. Germination was delayed for about one or two days. However, after 1-2 days the 24 hr treated seeds germinated and were free of smut. The germination rate was not reduced: at the 95% level, it was the same as the control. However, the seedling development in the treated seeds was just slightly behind that of the control group. As compared to a water control group, the treated seedlings were relatively free of fungal contamination. There was no smut spore development.
Because it is not practicable to soak seeds in a disease control agent before planting, it was decided to test use of Composition B with bentonite at concentrations of 0, 1, 5 and 10%. Bentonitie is used strictly as an inert carrier of B. To bentonite at 1 gram was added each of the ingredients to bring the concentration of B to these percentage levels. For instance, at the 10% level, 20 microliters each of propanoic acid, benzaldehyde, and isobutyl isobutyrate along with 50 mg of potassium isobutyrate was added to 1 gram of well-ground, powdery bentonite. The ingredients were thoroughly mixed in a mortar with pestle.
In a plastic bag, about 40 seeds were added to a bentonite/Composition B (10%) mixture. The contents were shaken. About 4 mg of freshly-harvested smut spores were then added. Bentonite adheres nicely to the seeds, as noted by microscopic observation. (As a control, bentonite powder without B was added to smut-infected seeds.) The seeds were held dry at room temperature for 5 days, after which 20-25 seeds from each group were carefully placed on to a Petri plate with PDA. Excess bentonite mixture that contained the smut spores used as inoculum was also placed on a portion of the plate. After incubation for 3 days, fungi grew in great proliferation on the control plate seeds, as well as on the control bentonite carrier. In contrast, the bentonite/B mixture and infected seeds treated with the bentonite/B mixture were relatively free of fungal growth.
An in planta experiment was conducted using oat seeds (20) and 1 g of powdered bentonite formulated with 10% Composition B (v/v/v w). The control was seeds with bentonite, alone. Each seed mixture was introduced to 4 mg of loose smut spores then incubated for 42 days* prior to planting. The seeds were placed into pots and were grown under greenhouse conditions with proper watering and fertilization for 5 weeks. The control plants were noticeably stunted after 5 weeks of growth, as compared to the treated plants.
The plants were held in their growth period for an additional 5-6 weeks under greenhouse conditions. At the end of 10-11 weeks from the time of planting they were evaluated for the development of any disease heads. This was done by excising each plant stem that was developing as a seed head. All stems having a seed head were evaluated. It is to be noted that the total number of seed heads in each group (control and treated) exceeds the total number of seeds planted because many of the plants tillered, and there was more than one seed head per plant. A total count of the diseased and healthy heads were made in the control and treated groups. As expected, there was more tillering—and more seed heads—in the treated group.
In the control group there were 26 heads counted, and 13 of these were diseased, for a 50% disease development rate. In contrast, the treated group had 33 heads, but only 3 heads were diseased, for a 9% disease development rate (i.e., a 91% control rate). The experiment was repeated and the typical stunting of the control plants was again observed. (*Disease control was less effective with less treatment time.)
As shown, Composition B, a representative composition of this invention, can be successfully used to treat seeds for the control of loose smut of oats, and other seed-borne diseases (e.g., reference experiments on mung beans). Typically, for a seed treatment, at least a 99% control rate for a seed borne disease is expected. Here, use of Composition B resulted in a 91% control rate of loose smut on oats. This rate can be improved by increasing the concentration of B on a carrier to, for instance, a level of 12 or 15%, to increase effectiveness. Again, an advantage of B is that all of its component ingredients are on the FDA-GRAS list and it, together with various other compositions of this invention, can be used for organic agricultural applications.
In accordance with other aspects of this invention, appropriate composition concentrations and time exposures can be developed for each plant type, with an awareness of possible adverse effects on germination.
While the results of this example show that B does have a controlling effect on loose smut of oats, other smuts including covered smuts of oats, barley etc. can be treated. Other seed borne diseases would also lend themselves for treatment with compositions of this invention, including diseases caused by damping off fungi.
Such compositions can also be used to treat soil-seed beds directly to reduce infection by seed borne diseases. The treating agent can be or incorporate a composition such as but not limited to B mixed with any number of carriers including bentonite or other inert carriers. Such a mixture can be co-planted with individual seeds or sprayed and mixed over the entire seed bed area.
While effective in reducing, eliminating or otherwise modulating disease incidence, various compositions of this invention—depending on relative component levels—can induce or cause some small necrotic spotting on more sensitive fruits (e.g., grapes). Without limitation to any one theory or mode of operation, it was thought that the propanoic acid level and/or composition pH may cause or induce the necrosis observed.
With reference to example 62a, a range of useful compositions and relative component amounts, of such compositions, were further assessed. A C4-C6 acid salt (e.g., potassium isobutyrate) was substituted for propanoic acid, maintaining a 7:2 (v/w) ratio of propanoic/isobutyrate to any other composition component (e.g., isobutyl isobutyrate or benzaldehyde). Accordingly, six compositions were formulated, as follows:
Evaluation, as observed:
All grapes in the control (untreated) container were seriously decayed. In contrast, fungal decay was not observed on any treated grape. However, under the conditions utilized, necrotic spotting can be modulated by compositions of the sort used in conjunction with treatments 3-6, preferably by those of the sort in treatments 4-5 and, optionally, 6—depending on the necrotic susceptibility of the fruit/vegetable treated. Without limitation, various compositions of this invention with a propanoic/isobutyrate ratio of about 4:3 to about 1:6 (e.g., a pH greater than about 4.4), preferably with a ratio of about 3:4 to about 2:5 (e.g., a pH of about 4.75 or greater) and, optionally, about 1:6, can effectively reduce or eliminate the incidence of necrosis.
The pH of any such composition is due primarily to the relative amounts of propanoic acid and a C4-C6 acid salt such as potassium isobutyrate and is largely independent of the presence and/or amount of C2-C5 acid ester(s), such as but not limited to isobutyl isobutyrate, and/or of C2-C8 aldehyde(s), such as but not limited to benzaldehyde. Accordingly, without limitation, compositions 3-6 having a pH greater than about 4.4 can be utilized with or without such an acid ester and/or aldehyde incorporated therein.
To further assess effectiveness of various non-limiting compositions of this invention, the minimum inhibitory concentration (MIC) of Composition B was determined against major fungal plant pathogens. Likewise, the MICs of Composition D (see, example 62) and Composition E (0.35 propanoic acid, 0.35 potassium isobutryate, 0.2 isobutyl isobutryate and 0.2 benzlaldehyde (v/w/v/v)) were determined. Each was made up to 1% in sterile water and diluted in twelve 1-milliliter well plates. Into each well was added 0.5 ml of potato dextrose broth along with water and either B, D or E to a final volume of 1 ml. Small 3 mm3 blocks of agar containing a specified pathogen were added, and MIC readings were made at 30 hr after the start of the experiment. The values in the Table 20 represent, respectively, the percent concentrations of B, D and E for 100% inhibition within 30 hr of incubation at room temperature.
Sclerotinia
Verticillum
Pythium
Aspergillus
Fusarium
Geotrichum
From the results observed, antimicrobial effect is maintained with partial substitution of an isobutyrate salt for propanoic acid. Likewise, as compared to Composition D, reduction in the number of components in Compositions B and E did not diminish biological activity. (Compare, e.g., the results of example 52.)
This application is a continuation in part of and claims priority to and the benefit of application Ser. No. 16/539,216 filed Aug. 13, 2019, which is a continuation of and claims priority to and the benefit of application Ser. No. 14/776,327 filed Sep. 14, 2015 and issued as U.S. Pat. No. 10,383,332 on Aug. 20, 2019, which claimed priority to and the benefit of International Application no. PCT/US2014/030657 filed Mar. 17, 2014, which claimed priority to and benefit of U.S. application Ser. No. 13/815,839 filed Mar. 15, 2013 and issued as U.S. Pat. No. 9,706,773 on Jul. 18, 2017—each of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14776327 | Sep 2015 | US |
| Child | 16539216 | US | |
| Parent | 13815839 | Mar 2013 | US |
| Child | 14776327 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16539216 | Aug 2019 | US |
| Child | 16778865 | US |
