BIODEGRADABLE CLEANING PRODUCTS, METHODS OF PRODUCTION, AND THEIR COMPOSITIONS

A biodegradable cleaning composition comprises one or more species of lactic acid bacteria, one or more species of Bacillus, water, and at least one additional element selected from the group consisting of at least one biodetergent, organic acid, diol, polyol, polymyxin, polysaccharide-lipid complex, fatty acid, monoterpene, sesquiterpene, preservative, essential oil, and carbon source. A biodegradable cleaning composition kit comprises at least one textile that has been treated with the biodegradable cleaning composition, wherein the treated textile is contained within an openable container.

TECHNICAL FIELD

The present disclosure relates to biodegradable cleaning compositions, methods of production, and methods of use. More particularly, the present disclosure comprises a microbial consortium in culture medium configured for cleaning use.

BACKGROUND

Scientific research and public opinion both increasingly value green technologies as replacements for traditional cleaning compositions. To achieve this end, beneficial microorganisms are used to obtain biochemicals through a controlled fermentation of natural ingredients. This process results in a composition comprising a consortium of viable beneficial microorganisms and their metabolites that provides cleaning properties.

SUMMARY

The present disclosure provides for one or more biodegradable cleaning compositions containing at least one (i) species of beneficial microorganism, (ii) organic acid, and (iii) carbon source. In one or more embodiments, the biodegradable cleaning composition may comprise (i) at least one species of microbe selected from the group consisting of lactic acid bacteria andBacillus, (ii) at least one carboxylic acid, (iii) at least one carbon source, and (iv) one or more added elements such as but not limited to an essential oil, monoterpene, sesquiterpene, carboxylic acid, fatty acid, and/or polyol.

The present disclosure also provides for one or more methods of using one or more biodegradable cleaning compositions of the present disclosure, generally comprising contacting a surface with a composition made according to any embodiment described herein. One illustrative and non-limiting example of a surface may comprise a countertop. One or more biodegradable cleaning compositions may be useful to achieve other beneficial outcomes as well.

DETAILED DESCRIPTION

The present biodegradable cleaning composition may comprise at least one species of beneficial microorganism, at least one organic acid, at least one carbon source, and water. In some embodiments, the biodegradable cleaning composition may comprise one or more species of lactic acid bacteria, one or more species ofBacillus, water, and at least one additional element selected from the group consisting of at least one essential oil, carbon source, biodetergent, organic acid, diol, polyol, polymyxin, polysaccharide-lipid complex, fatty acid, monoterpene, sesquiterpene, ketone, hydrocarbon, aldehyde, polymer, and gas.

In some embodiments, the present invention may comprise a kit comprising an individual use packet containing a textile that has been soaked in the biodegradable cleaning composition. In some embodiments, the present invention may comprise a kit comprising a dispenser that contains multiple textiles that have been soaked with the biodegradable cleaning composition.

In some embodiments, the at least one species of lactic acid bacteria may be one or more species ofCarnobacteriumsp.Enterococcussp.Lactobacillussp.Lactococcussp.Leuconostocsp.Oenococcussp.Streptococcussp.Tetragenococcussp.Vagococcussp. andWeissellasp.

In some embodiments, the at least one species ofBacillusmay be selected from the group consisting ofBacillus licheniformis, Bacillus coagulans, andBacillus subtilis.

In some embodiments, the microorganism consortium in culture medium may produce one or more metabolites. These metabolites, in some embodiments, may be characterized by strong polar groups, mainly anionic, and active functional radicals that enable them to combine with other organic molecules. In some embodiments, the metabolites may provide high reactivity to combine other molecules through multiple functional radicals. The metabolites, in some embodiments, may comprise molecules that can disseminate into small spaces, such as between fibers or into fine cracks on a surface, such as a countertop. The metabolites, in some embodiments, may possess bactericidal-like properties due to the presence of one or more carboxylic acids. The metabolites, in some embodiments, may possess hydrotropic properties that provide hydration and increase solubility of the substances to which they combine.

In some embodiments, the biodegradable cleaning composition may be prepared by fermenting a culture medium for a period of time. In some embodiments, the fermentation period may comprise at least 5 days. In preferred embodiments, the fermentation period may comprise 14 days or more.

In some embodiments, following the fermentation period, the fermented product may be decanted from a fermentation tank via at least one centrifuge. In some embodiments, the centrifuge may be a solid-liquid separation centrifuge. The solid-liquid separation centrifuge, in some embodiments, may comprise a bowl centrifuge configured to run at 10,000 rpm to 15,000 rpm for a gravitational force equivalent (g-force) of 12,400 g to 15,000 g, or in some embodiments at 22,000-23,000 rpm. In some embodiments, the fermented product may be passed through a filter prior to introduction into the centrifuge. In some embodiments, the filter may comprise a 30-micron filter. In some embodiments, after passing through the filter, the fermented product may be fed into the centrifuge at a material feed rate of 150 cc/minute. The centrifuge may spin the base material at 22,000 to 23,000 rpm for one or more minutes. The biodegradable cleaning composition may then be extracted from the centrifuge.

As used herein, the term “additional element” refers to any element named in reference to the term “added element,” whether that element is a metabolite of any microorganism found in the biodegradable cleaning composition or a metabolite produced by a microorganism of another source, or whether the element is produced via another process not involving a microorganism and is then added to the biodegradable cleaning composition at any step or phase, including but not limited to any day or stage of fermentation or to its finished form or state. Generally, it is contemplated that one or more microbial species of the biodegradable cleaning composition may produce the one or more added elements that may comprise part of the biodegradable cleaning composition. In other embodiments, however, the one or more added elements of the biodegradable cleaning composition may be produced independently of the biodetergent cleaning composition and may be added to the biodegradable cleaning composition. It is contemplated, therefore, that any added element comprising part of the biodegradable cleaning composition may have been produced as a metabolite by a microbe that is itself part of the biodegradable cleaning composition, may have been produced by another means and added to the biodegradable cleaning composition, or both.

Similarly, as used herein, the term “biodetergent” refers to any detergent produced as a metabolite by a microorganism, whether the microorganism is a part of the biodegradable cleaning composition or of another source. “Detergent” means a detergent not produced by a microorganism. Similarly, any molecule produced by a microorganism may be referred to herein as a biomolecule. Generally, it is contemplated that one or more microbial species of the biodegradable cleaning composition may produce the one or more biodetergents that may comprise part of the biodegradable cleaning composition. In other embodiments, the one or more biodetergents of the biodegradable cleaning composition may be produced independently of the biodetergent cleaning composition and may be added to the biodegradable cleaning composition. It is contemplated, therefore, that any biodetergent comprising part of the biodegradable cleaning composition may have been produced as a metabolite by a microbe that is itself part of the biodegradable cleaning composition, may have been produced by another means and added to the biodegradable cleaning composition, or both.

In some embodiments, the biodegradable cleaning composition may comprise one or more polymyxins, such as but not limited to polymyxin A, polymyxin B, polymyxin B1, polymyxin B nonapeptide, polymyxin C, polymyxin D, and polymyxin E (colistin), and other cationic cyclic decapeptides.

In some embodiments, the biodegradable cleaning composition may comprise one or more polysaccharide-lipid complexes, and polysaccharide-lipid-protein complexes, such as but not limited to lipopolysaccharides, lipooligosaccharides, and endotoxins produced by a microbe selected from the group comprising but not limited toE. coliO26,E. coliEH-100,E. coli0111,E. coli055,E. coli0127,E. coli0128,E. coliF-583,E. coliJ5,E. coliK-235,Pseudomonas aeroginosa10,Salmonella, Salmonella enteritidis, Salmonella minnesota, Salmonella minnesotastrain Re595,Salmonella typhimurium, Salmonella typhimuriumstrain SL1181,Salmonella typhimuriumstrain TV119,Salmonella typhosa, andSerratia marcescens.

In embodiments containing one or more preservatives, by way of non-limiting illustration, the biodegradable cleaning composition may comprise one or more preservatives in a ratio of from about 0.01-3.0 wt/wt %. Preferably, the biodegradable cleaning composition may comprise one or more preservatives in a ratio of from about 0.1-0.3 wt/wt %.

In embodiments of the biodegradable cleaning composition comprising one or more preservatives, the biodegradable cleaning composition containing one or more preservatives may demonstrate a novel and unexpected antimicrobial effect. At least one antimicrobial effect may comprise, but it not limited to, the inhibition or elimination of pathogens on a surface without the inhibition or elimination of one or more microorganisms comprising part of the biodegradable cleaning composition. Data confirming this premise is presented as Example 3. In preferred embodiments, the preservative may comprise potassium sorbate in a ratio of from about 0.01-0.3 wt/wt % of the biodegradable cleaning composition. In other preferred embodiments, the preservative may comprise potassium sorbate in a ratio of from about 0.01-0.1 wt/wt % of the biodegradable cleaning composition. However, any ratio described herein is contemplated. In other preferred embodiments, the preservative may comprise citric acid or organic citric acid in a ratio of from about 0.01-0.3 wt/wt % of the biodegradable cleaning composition. In other preferred embodiments, the preservative may comprise potassium sorbate in a ratio of from about 0.1-0.5 wt/wt % of the biodegradable cleaning composition. However, any ratio described herein is contemplated.

In some embodiments, the biodegradable cleaning composition containing one or more preservatives may inhibit or eliminate pathogens such as, but not limited to,S. typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, andEscherichia coli. Additional pathogenic microorganisms that may be inhibited or eliminated by the biodegradable cleaning composition can be found inA Clinician's Dictionary of Pathogenic Microorganismsby James H. Jorgensen, Michael A. Pfellerauthors, ASM Press, Washington, DC, ISBN: 1-55581-280-5.

In some embodiments, the biodegradable cleaning composition may also comprise one or more essential oils (or “natural oils”) that may, in some embodiments, serve as a deodorant, a deodorizer, an insect repellant, an insecticide, an adjuvant that enhances one or more of the biodegradable cleaning composition's other properties, or as a fertilizer or biostimulant. Such essential oils or natural oils may include, without limitation, African lemon bush (Lippia javanica) oil, anise oil, bay oil, bergamot oil,boroniaoil, canola oil, carrot oil,cassiaoil, catnip oil, cedarwood oil, chamomile oil, cilantro oil, cinnamon oil, citronella oil, clary sage oil, clove oil, coriander oil, cypress oil,eucalyptusoil,galbanumoil, garlic oil, ginger oil, geranium oil, grapefruit oil, hazelnut oil, jasmine oil, jojoba oil, lavender oil, lavandin oil, lemon oil, lemongrass oil, lime oil, mandarin oil, nutmeg oil, orange oil, palma rosa oil, patchouli oil, pine oil, Peru balsams, peppermint oil, rosemary oil, rosewood oil, sage oil, sandalwood oil, spear mint oil, star anise oil, tea tree oil, tangerine oil, thyme oil, tolu balsam oil,verbenaoil, white clover oil, ylang ylang oil, and combinations thereof. Essential oils may be organic or not organic. Preferably, the essential oil is provided in an organic form. In preferred embodiments, the essential oil is present in an organic form.

In some embodiments, the at least one added element may further comprise a hydrocarbon, such as but not limited to cyclohexane, cyclohexene, 3-acetoxy-4-(1-hydroxy-1-methylethyl)-1-methyl, cycloehexane, an aldehyde such as but not limited to 2-furaldehyde (furfural), a ketone such as but not limited to sulcatone, nonanone, or 4-nonanone, tagalose, D-(−)-tagalose, D-(+)-tagalose, mannopyranose, b-D-(+)-mannopyranose, b-D-(−)-mannopyranose, sulctone, 2,3-dehydro-1,8-cineole, a-phellandren-8-ol, 2,6-octadien-1-ol, 3,7-dimethyl-acetate, caryophyllene, g-cadinene, cadina-1(10),4-diene, carophyllene oxide, [(2-ethyl-5-methylfuran-3,4-diyl)bis(oxy)]bis(trimethylsilane), a polymer such as siloxane or pentasiloxane, or a gas such as silane or [(2-Ethyl-5-methylfuran-3,4-diyl)bis(oxy)]bis(trimethylsilane).

The biodegradable cleaning composition may further comprise one or more carbon sources, including rum, molasses, glucose, starch, cellulose, fructose, sucrose, or the like. In one embodiment, the carbon source is plant material such as silage. In another embodiment, the carbon source is a plant material from the grass family Poaceae. In some embodiments, the carbon source may comprise a relatively unrefined plant material, such as silage, stover, chaff, grass, stalks, leaves, and the like. In other embodiments, the carbon source comprises a more refined plant material, such as flour, syrup, molasses, or the like. In another embodiment, the carbon source comprises purified or semi-purified organic molecules, such as protein, fat, fatty acids, carbohydrates, or the like. In a more specific embodiment, the carbon source comprises a bran from grain, such as, e.g., rice bran, or a syrup or molasses from sugar cane. Further examples of suitable carbon sources include, without limitation, peptone, yeast extract, amino acids, other sugars such as arabinose, mannose, glucosamine, maltose, sugar cane, molasses, rum, and the like; salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, methylmaleic acid, pyruvic acid, malonic acid and the like; alcohols such as ethanol, glycerol, glycerol trilaurate, and the like; or oil or fat such as soybean oil, rice bran oil, olive oil, corn oil, and sesame oil. The amount of the carbon source added varies according to the kind of carbon source and is typically between 1 to 100 grams per liter of medium. The weight fraction of the carbon source in the biodegradable cleaning composition may be about 98% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the biodegradable cleaning composition. Preferably, molasses is contained in the medium as a carbon source, at a concentration of about 2 to 20% (w/v). More preferably, the molasses is at a concentration of about 4 to 6% (w/v).

In some embodiments, the biodegradable cleaning composition may be applied to a textile or textile-derived products, such as but not limited to a towel, wipe, gauze roll, gauze pad, cloth, glove, handkerchief, washcloth, mophead, microfiber cloth, or other porous or nonporous item configured to apply the biodegradable cleaning composition to a surface. In some embodiments, the textile may be composed of lyocell, rayon, cotton polyester, polypropylene, or more generally of woven or nonwoven materials, or synthetic or nonsynthetic materials. Any commercially available textile known to be used in cleaning wipes is contemplated herein.

In embodiments providing for the use of the biodegradable cleaning composition on a textile, the biodegradable cleaning composition may be applied to the textile according to at least one ratio. In such embodiments, the ratio my comprise 0.01 ml per sheet to 30 ml per sheet, 1 ml per sheet to 20 ml per sheet, 5 ml per sheet to 15 ml per sheet, 1 ml per sheet, 1.5 ml per sheet, 2 ml per sheet, 2.5 ml per sheet, 3 ml per sheet, 3.5 ml per sheet, 4 ml per sheet, 4.5 ml per sheet, 5 ml per sheet, 5.5 ml per sheet, 6 ml per sheet, 6.5 ml per sheet, 7 ml per sheet, 7.5 ml per sheet, 8 ml per sheet, 8.5 ml per sheet, 9 ml per sheet, 9.5 ml per sheet, 10 ml per sheet, 10.5 ml per sheet, 11 ml per sheet, 11.5 ml per sheet, 12 ml per sheet, 12.5 ml per sheet, 13 ml per sheet, 13.5 ml per sheet, 14 ml per sheet, 14.5 ml per sheet, 15 ml per sheet, 15.5 ml per sheet, 16 ml per sheet, 16.5 ml per sheet, 17 ml per sheet, 17.5 ml per sheet, 18 ml per sheet, 18.5 ml per sheet, 19 ml per sheet, 19.5 ml per sheet, 20 ml per sheet, 20.5 ml per sheet, and greater than 20.5 ml per sheet. In some embodiments, the ratio is from 6.67 ml per sheet to 11.67 ml per sheet. All of the above ratio ranges are contemplated.

In some embodiments, one or more compounds of the biodegradable cleaning composition may be produced by fermentation of selected probiotic strains in a selected medium under defined fermentation conditions; wherein final product has a pH value of <4.0 with activity in pH range from 2 to 12, percent total acidity (Lactic Acid meq) between about 0.6 and about 3.2 E24 values-between about 50% and about 90%; and surface tension between about 20 dyne/cm and about 38 dyne/cm.

In some embodiments, methods of manufacturing the biodegradable cleaning composition may comprise adding one or more stabilizers, microbial inoculants, antioxidants, or preservatives to the starting probiotic culture, the fermented product, or the biodegradable cleaning composition product. In some embodiments, one or more elements named elsewhere herein, such as but not limited to one or more biodetergents, fatty acids, polyethylene glycols, polyols, diols, polysaccharide-lipid complexes, may be used as one or more stabilizers. In some embodiments, one or more organic acids, monoterpenes, sesquiterpenes, essential oils, diols, or any other element named herein may be used as one or more preservatives. In some embodiments, one or more organic acids, diols, polyols, monoterpenes, sesquiterpenes, essential oils, biodetergents, or any other element named herein may be used as one or more antioxidants. In some embodiments, one or more stabilizers, microbial inoculants, antioxidants, or preservatives may comprise one or more element of the biodegradable cleaning composition.

Trial of at least one embodiment of the biodegradable cleaning composition indicate that the use of a centrifuge as described herein may provide for the enhancement of product consistency and efficacy. Specifically, the results tend to show higher consistency in product clarity by using the centrifuge. The advantage to this unique and unpredictable discovery lies in the fact that the centrifuge lowers turbidity levels in the product, which increases efficacy as measured by one or more cleaning efficiency tests. Accordingly, the novel and nonobvious use of the centrifuge as described herein provides enhanced benefits to a user.

In some embodiments, the biodegradable cleaning composition may be in a concentrated or diluted form. In some embodiments, 5-99% wt/wt % of the biodegradable cleaning may comprise water. In some embodiments, 20-80% wt/wt % of the biodegradable cleaning composition may comprise water. In some embodiments, 30-70% wt/wt % of the biodegradable cleaning composition may comprise water. In some embodiments, 75-97% wt/wt % of the biodegradable cleaning composition may comprise water. In some preferred embodiments, 80-85% wt/wt % of the biodegradable cleaning composition may comprise water. In other preferred embodiments, 90-95% wt/wt % of the biodegradable cleaning composition may comprise water.

In some embodiments of the present invention, the biodegradable cleaning compositions may comprise hydrotropes that possess the ability to increase solubility of sparingly soluble molecules in water. They may include some hydrophilic and lipophilic fractions, like typical surfactants, but they may also contain a very small hydrophobic fraction that does not allow the formation of aggregates such as micelles. The final biodegradable composition used in place of or in addition to conventional cleaning products or detergents may comprise not only biochemical/metabolite fermentation broth, but also viable probiotic microorganisms with a defined profile based on metagenomics and metabolomics analysis (ie., fingerprint).

In some embodiments, the biodegradable cleaning composition may provide a metagemonics profile characterized by percent Relative Richness of microorganisms such as one or more lactic acid bacteria and one or moreBacillussp. as presented in more detail herein, in ranges from about 1 to about 98%. More preferably, the range is from about 20% to about 40%.

In some embodiments, the biodegradable cleaning composition may provide a metabolomics profile characterized by one or more untargeted metabolomics analyses provided in the experiments section herein. The untargeted metabolomics analysis may indicate, without limitation, one or more metabolites produced by one or more microorganisms of the present biodegradable cleaning composition, which may include but are not required to include, without limitation, one or more biodetergents, organic acids, diols, polyols, polymyxins, polysaccharide-lipid complexes, fatty acids, monoterpenes or monoterpenoids, sesquiterpenes or sesquiterpenoids, preservatives, or other compound referred to herein as a second metabolite.

In some embodiments, the biodegradable cleaning composition contains Lactic Acid Bacteria counts of from about 1.0E+1 to about 1.0E+6. Preferably, the biodegradable cleaning composition includes Lactic Acid Bacteria counts of from about 1.0E+5 to about 8.0E+5 CFU per milliliter. More preferably, the biodegradable cleaning composition includes Lactic Acid Bacteria counts of from about 2.50E+5 to about 6.0E+5 CFU per milliliter. Most preferably, the biodegradable cleaning composition includes a total number of microorganisms of about 3.0E+5 CFU per milliliter.

In some embodiments, the biodegradable cleaning composition containsBacilluscounts of from about 1.0E+1 to about 1.0E+6. Preferably, the biodegradable cleaning composition includesBacilluscounts of from about 1.0E+5 to about 8.0E+5 CFU per milliliter. More preferably, the biodegradable cleaning composition includesBacilluscounts of from about 2.50E+5 to about 6.0E+5 CFU per milliliter. Most preferably, the biodegradable cleaning composition includesBacilluscounts from about 3.0×10{circumflex over ( )}5 to about 4.8×10{circumflex over ( )}5 CFU per milliliter. However, any range discussed herein or described in an example may constitute a preferred amount or range.

Additionally, in some embodiments, the biodegradable cleaning composition may provide viable and active probiotics at the following minimums: lactic acid bacteria minimum 8.9×10{circumflex over ( )}4 cfu/g andBacillusminimum 3.2×10{circumflex over ( )}4 cfu/g. The biodegradable cleaning composition may also have a pH of <3.5 and be free of detectable mold and coliforms. The biodegradable cleaning composition may provide for a surface tension minimum of 32 dyne/cm. The biodegradable cleaning composition may have a % Titratable Acidity of from about 0.03 to about 0.4%.

In embodiments wherein the biodegradable cleaning composition has been applied to a textile, the biodegradable cleaning composition may have a pH of <3.2 as well as viable and active probiotics (LAB minimum 1.0×10{circumflex over ( )}5 cfu/g andBacillusminimum 1.0×10{circumflex over ( )}5 cfu/g).

Using Test Method ASTM D4488-A5, the biodegradable cleaning composition may provide a cleaning efficacy (CE) of 80% (minimum) 7 days after dilution 87% CE and 80% CE (minimum) 21 days after dilution.

Moreover, in some embodiments, and as certain examples provided herein tend to show, the beneficial microorganisms of the biodegradable cleaning composition may form spores on surfaces, and the spores may re-germinate. In some embodiments, the spores may regerminate beginning less than 24 hours of application. In some embodiments, the spores may regerminate beginning at 24 hours of application. In some embodiments, the spores may regerminate beginning after 24 hours of application. Example 3 shows results of experimental data supporting this assertion.

In some embodiments, therefore, embodiments of the biodegradable cleaning composition provide several beneficial aspects. For example, the biodegradable cleaning composition provides live, viable probiotics and probiotic-produced biosurfactants and organic acids that give it triple microscopic cleaning capability.

In some embodiments, the biodegradable cleaning composition is essentially free of purple sulfur bacteria. In another preferred embodiment, the biodegradable cleaning composition contains purple sulfur bacteria. In some embodiments, the biodegradable cleaning composition contains purple sulfur bacteria at a level of from about 1.0E+1 to about 1.0E+5, and preferably, from about between 1.0E+1 to 1.0E+4.

In some embodiments, the biodegradable cleaning composition is essentially free of pathogenic or putrefactive microorganisms. Preferably, the biodegradable cleaning composition is essentially free of pathogenic mold and total coliform. Most preferably, pathogenic mold and total coliform is undetectable in the biodegradable cleaning composition.

In some embodiments, one or more embodiments of the biodegradable cleaning composition may be formulated as a counter-cleaner solution, an all-purpose cleaning solution, a floor cleaning solution, a floor cleaning concentrate, a probiotic floor cleaning concentrate, an air freshener solution, a fabric refresher solution, an air and fabric freshener solution, a window cleaning solution, or any solution used to treat or clean a surface or the air.

It is contemplated that where two or more microorganisms are present within one or more embodiments of the biodegradable cleaning composition, the microorganisms may be co-cultured. The microorganisms may be propagated by methods known in the art. For example, the microorganisms may be propagated in a liquid medium under anaerobic or aerobic conditions. Suitable liquid mediums used for growing microorganisms include those known in the art. It is contemplated that, in some embodiments, the one or more strains of useful microorganisms described herein may be co-cultured under laboratory conditions in any combination before being transferred to one or more fermentation tanks. In some embodiments, the one or more strains of useful microorganisms may be co-cultured partly under laboratory conditions, then transferred to one or more fermentation tanks, where the co-culture process may continue. In other embodiments, the one or more strains of useful microorganisms may be co-cultured within one or more fermentation tanks.

In some embodiments, the one or more microbial organisms of the microorganism consortium may include, but are not limited to,Bacillus licheniformisWeigmann Chester ATCC 25972 orBacillus thuringiensisBerliner ATCC 700872.

In some embodiments, the one or more microbial organisms of the microorganism consortium may include, but are not limited to,Bacillus weihenstephanensisLechner et al. ATCC 12826,Bacillus weihenstephanensisbacteriophage ATCC 12826-B2, 31293, 31429, 31292, orBacillussp. ATCC 13062, P7 ATCC 75237.

In some embodiments whereinEnterococcus lactiscomprises one or more microbial organisms of the microorganism consortium, illustrative strains may include, but are not limited to,Enterococcus lactisBT159 T,Enterococcus lactisCCM 8412,Enterococcus lactisDSM 23655Enterococcus lactisLMG 25958,Enterococcus lactisMorandi BT159, or any other species ofEnterococcus lactis.

In some embodiments whereinLactobacillus parafarraginiscomprises one or more microbial organisms of the microorganism consortium, illustrative strains may include, but are not limited to,Lactobacillus parafarraginisNCIMB 15108 orLactobacillus parafarraginisNCIMB 702943.

In some embodiments, the one or more microbial organisms of the microorganism consortium may include, but are not limited to,Lactobacillus rapiDSM-19907,Lactobacillus rapiType Strain JCM 15042 NRIC 0743,Leuconostoc rapiDSM-27776, orLeuconostoc rapiType Strain LMG 27676.

In some embodiments, the biodegradable cleaning composition includes a total number of microorganisms of about 1 to about 1 million colony forming units (CFU) per milliliter. Preferably, the biodegradable cleaning composition includes a total number of microorganisms of about 100,000 to about 800,000 CFU per milliliter. More preferably, the biodegradable cleaning composition includes a total number of microorganisms of about 250,000 to about 600,000 CFU per milliliter. Most preferably, the biodegradable cleaning composition includes a total number of microorganisms of about 300,000 CFU per milliliter.

In some embodiments, the present composition contains a first mixture of live microorganisms in the at least one microorganism consortium in culture medium, wherein the live microorganisms are present at a collective “low titer”, and one or more additional non-pathogenic gram-positive bacteria of the Bacilli family that are capable of fermentation at a “high titer” for each additional individual species/strain of bacteria. By collective titer, what is meant is the individual titers of each of the first live microorganism adds up to the collective titer. To use an example of live lactic acid bacteria (LAB), if the (LAB) consists of 15,000 cfu/mL of aBacillussp. 12,000 cfu/mL of aLactobacillussp. and 10,000 cfu/mL of aStreptococcussp. then the collective titer is 37,000 cfu/mL of LAB. In some embodiments, “low titer” is <300,000 cfu/mL, about 0.001-299,999 cfu/mL, about 1,000-250,000 cfu/mL, 5,000-200,000 cfu/mL, 10,000-150,000 cfu/mL, 15,000-100,000 cfu/mL, 20,000-75,000 cfu/mL, 25,000-60,000 cfu/mL, 30,000-55,000 cfu/mL, about 10,000 cfu/mL, about 11,000 cfu/mL, about 12,000 cfu/mL, about 13,000 cfu/mL, about 14,000 cfu/mL, about 15,000 cfu/mL, about 16,000 cfu/mL, about 17,000 cfu/mL, about 18,000 cfu/mL, about 19,000 cfu/mL, about 20,000 cfu/mL, about 21,000 cfu/mL, about 22,000 cfu/mL, about 23,000 cfu/mL, about 24,000 cfu/mL, about 25,000 cfu/mL, about 30,000 cfu/mL, about 35,000 cfu/mL, about 40,000 cfu/mL, about 45,000 cfu/mL, about 50,000 cfu/mL, about 55,000 cfu/mL, about 60,000 cfu/mL, about 65,000 cfu/mL, about 70,000 cfu/mL, about 75,000 cfu/mL, about 80,000 cfu/mL, about 85,000 cfu/mL, about 90,000 cfu/mL, about 95,000 cfu/mL, about 100,000 cfu/mL, about 105,000 cfu/mL, about 110,000 cfu/mL, about 115,000 cfu/mL, about 120,000 cfu/mL, about 125,000 cfu/mL, about 150,000 cfu/mL, about 175,000 cfu/mL, about 200,000 cfu/mL, about 225,000 cfu/mL, about 250,000 cfu/mL, about 275,000 cfu/mL, or about 299,000 cfu/mL.

In those embodiments in which the present composition is in a dry form, the liquid present compositions described herein are dried. “Dry form” refers to a composition containing ≤15% water by weight, ≤14% water by weight, ≤13% water by weight, ≤12% water by weight, ≤11% water by weight, ≤10% water by weight, ≤9% water by weight, ≤8% water by weight, ≤7% water by weight, ≤6% water by weight, ≤5% water by weight, ≤4% water by weight, ≤3% water by weight, ≤2% water by weight, ≤1% water by weight, 1%-5% water by weight, 2%-6% water by weight, about 3% water by weight, about 4% water by weight, about 5% water by weight, about 6% water by weight, about 7% water by weight, or about 8% water by weight.

The biodegradable cleaning compositions of the invention may be in liquid or dry form. The biodegradable cleaning composition may comprise an aqueous suspension. This aqueous suspension may be provided as a concentrated stock solution which is diluted prior to application or as a diluted solution ready-to-use.

Also, the biodegradable cleaning composition may be a wettable powder, granules, dust, pellet, or colloidal concentrate. Such dry forms may be formulated to dissolve immediately upon wetting with water or suitable solvent system, or dissolve in a controlled-release, sustained-release, or other time-dependent manner. Also, the biodegradable cleaning composition may be in a dry form that does not depend upon wetting or dissolving to be effective.

The biodegradable cleaning compositions may additionally be provided in a formulation capable of spray. Spray compositions may include surfactants or biodetergents, emulsifiers, and wetting agents. The spray may be a liquid or an aerosol. The biodegradable cleaning compositions of the present invention are stable under various conditions as a liquid or dry form. Preferably, the biodegradable cleaning compositions of the present invention are stable at room temperature.

In some embodiments, the dry form of the present composition is produced by evaporation, spray-drying, lyophilization, or the like. In some embodiments, the dry present composition is encapsulated to promote the stability and viability of the microbes over time and under varying temperature conditions. In some embodiments, the dry present composition is divided into micron scale particles that are subsequently coated with biocompatible polymers, such as polyethylene glycol (PEG), chitin, dextrin, polylactic glycolic acid copolymer (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), or the like.

In still another embodiment, the excipient may comprise a buffering agent. Representative examples of suitable buffering agents include, but are not limited to, MOPS, HEPES, TAPS, Bicine, Tricine, TES, PIPES, MES, Tris buffers or buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).

In yet another embodiment, the excipient may include a dispersion enhancer. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isomorphous silicate, and microcrystalline cellulose.

In a further embodiment, the excipient may include a lubricant. Non-limiting examples of suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate, or stearic acid.

In still another embodiment, it may be desirable to provide a coloring agent. Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C). Such colors may include but not be limited to red, yellow, green, blue, white, black, brown, purple, orange, any shade thereof, and any other color produced according to the above-named color formats.

The weight fraction of the excipient(s) in the combination may be about 98% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the combination.

The biodegradable cleaning compositions of the present disclosure are stable under various conditions as a liquid or dry form. Preferably, the biodegradable cleaning compositions of the present disclosure are stable at room temperature.

Methods of Manufacture

In some embodiments, the biodegradable cleaning composition may be manufactured by at least one method, in some embodiments comprising one or more steps of: (a) obtaining a starting composition comprising at least one species of microbe selected from the group consisting of at least one species of lactic acid bacteria and at least oneBacillusspecies; at least one organic acid; at least one carbon source; and dechlorinated water; (b) beginning a fermentation period, wherein the probiotic starting culture is fermented in at least one fermentation tank for at least one fermentation period; (c) adding at least one additional element to the probiotic starting culture, wherein, in some embodiments the at least one additional element may comprise an essential oil; (d) allowing the probiotic starting culture and the at least one additional element to ferment into a fermented product; (e) ending the fermentation period; and (f) decanting the fermented product using a centrifuge into a biodegradable cleaning composition.

In one or more embodiments of the present disclosure, the biodegradable cleaning composition may be prepared partly or entirely within one or more fermentation tanks, or bioreactor as provided in more detail below. In some embodiments, less than ten fermentation tanks may be provided. In some embodiments, less than twenty fermentation tanks may be provided. In some embodiments, more than twenty fermentation tanks may be provided. In some embodiments, one or more fermentation tank may be a primary fermentation tank. In one or more embodiments, one or more fermentation tanks may be a horizontal fermentation tank. In one or more embodiments, one or more fermentation tanks may be a vertical fermentation tank.

In some embodiments, one or more primary fermentation tanks may provide for a maximum capacity of at least 500 liters. In some embodiments, one or more primary fermentation tanks may provide for a maximum capacity of at least 5000 liters. In some embodiments, one or more primary fermentation tanks may hold at least 2006.40 liters. In some embodiments, one or more primary fermentation tanks may hold at least 5639.65 liters. In some embodiments, one or more primary fermentation tanks may hold at least 5678.00 liters. In some embodiments, one or more primary fermentation tanks may hold at least 11440.00 liters.

In some embodiments, one or more horizontal fermentation tanks may provide for a maximum capacity of at least 500 liters. In some embodiments, one or more horizontal fermentation tanks may hold at least 2006.40 liters. In some embodiments, one or more horizontal fermentation tanks may provide for a maximum capacity of at least 5000 liters. In some embodiments, one or more horizontal fermentation tanks may hold at least 5639.65 liters. In some embodiments, one or more horizontal fermentation tanks may hold at least 5678.00 liters. In some embodiments, one or more horizontal fermentation tanks may hold at least 11440.00 liters.

In some embodiments, one or more vertical fermentation tanks may provide for a maximum capacity of at least 500 liters. In some embodiments, one or more vertical fermentation tanks may hold at least 2006.40 liters. In some embodiments, one or more vertical fermentation tanks may provide for a maximum capacity of at least 5000 liters. In some embodiments, one or more vertical fermentation tanks may hold at least 5639.65 liters. In some embodiments, one or more vertical fermentation tanks may hold at least 5678.00 liters. In some embodiments, one or more vertical fermentation tanks may hold at least 11440.00 liters.

In one or more embodiments, the present disclosure may comprise five primary fermentation tanks having capacities of at least 5639.65 liters, two primary fermentation tanks having capacities of at least 11440.00 liters, three horizontal fermentation tanks having capacities of at least 5678.00 liters, and three vertical fermentation tanks having capacities of at least 2006.40 liters.

The microorganism consortium may, as provided elsewhere herein, be co-cultured partly or entirely, co-cultured prior to addition to a fermentation tank, co-cultured partly before and partly after addition to a fermentation tank, or co-cultured within a fermentation tank.

In some embodiments, the fermentation tank may comprise a bioreactor. Any and all details applicable to fermentation tanks may apply to one or more bioreactors.

The bioreactor of the present disclosure may, in some embodiments, comprise a batch reactor, a semi-batch reactor, a fed batch reactor, a photobioreactor, a continuous reactor, or another type of bioreactor. The bioreactor's exterior and/or interior may be comprised of stainless steel, copper, plastic, or equivalent material(s). The bioreactor may provide for one or more temperature control elements, one or more aeration elements, one or more agitation elements, one or more baffles, one or more spargers, one or more jackets, one or more scrubbers, and at least one inflow/outflow portal. The bioreactors may comprise an immobilized cell bioreactor, such as a large-scale immobilized cell bioreactor. The bioreactor(s) may be adiabatic or non-adiabatic. The bioreactor may comprise a gas-phase bioreactor, which may be a one-step, two-step, or three- or more step gas-phase bioreactor. The bioreactor may comprise a chemostat or may support chemostatic activity or usage. The bioreactor may comprise a continuous stirred tank reactor. The bioreactor may comprise a plug flow reactor. The bioreactor may be pressurized. When two or more bioreactors are used, the bioreactors may be connected in series, in parallel, or both in series and in parallel. The bioreactor may be used in a fed-batch manner or for semi-continuous fermenter operation. The bioreactor may comprise a moving bed biofilm reactor, packed-bed, a fibrous-bed, or a membrane bioreactor.

In some embodiments, the temperature control element comprises a water jacket inside the bioreactor through which water may flow changing the temperature of the tank and thereby reducing or increasing the temperature of the bioreactor's contents. The temperature control element may comprise one or more coils that may surround the bioreactor's tank, be placed underneath the tank's base, around the top of the tank, or at one or more location.

The bioreactor agitation elements may, in some embodiments, comprise a mixer mounted to a bioreactor top element, a circulation system wherein the contents of the bioreactor are siphoned off from the bottom of the bioreactor and reintroduced at the top of the bioreactor's interior, shaking the bioreactor, shaking the contents of the bioreactor on a platform within the bioreactor, or other known methods of agitation. The agitation element may comprise a gas dispersing agitator.

Additionally, in some embodiments, the agitation element may comprise, in some embodiments, one or more anchor agitator, propeller agitator, radial propeller agitator, turbine agitator, umbrella agitator, paddle(s) agitator, flat blade agitator, ruvastar cyclo agitator, spiral propeller blade agitator, high shear homogenizer, open blade agitator, or other agitation element located inside a bioreactor. In some embodiments, agitation may comprise cycling the fermented product inside a bioreactor, or by passing air upwards through a fermented product inside a bioreactor.

The bioreactor's scrubber may comprise a wet scrubbing system, a dry scrubbing system, or both. The present disclosure's outgassing apparatus may comprise a scrubber or scrubbing system.

In some embodiments, the probiotic starting culture, fermented product, or other element of the present disclosure having a microorganism consortium (each a “fermentable element”) may be fermented for a time prior to its insertion into a bioreactor or before its combination with one or more elements.

In some embodiments, the probiotic starting culture may be fermented for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more days. Preferably, the probiotic starting culture is fermented for at least about 15 to about 23 days. More preferably, the probiotic starting culture is fermented for at least 21 days. In some embodiments, the fermentation time for the probiotic starting culture prior to its insertion into a tank or combination with one or more elements is at least 15 days. The fermentation may take place in a tank, a bioreactor, or any other vessel.

In some embodiments, another fermentable element may be fermented for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more days. Preferably, the other fermentable element is fermented for at least about 15 to about 23 days. More preferably, the other fermentable element is fermented for at least 21 days. In some embodiments, the fermentation time for the other fermentable element prior to its insertion into a tank or combination with one or more elements is at least 15 days. The fermentation may take place in a tank, a bioreactor, or any other vessel.

The present disclosure may provide that one or more volumes of the probiotic starting culture be added to one or more primary fermentation tanks. Without limitation, less than 10 liters, less than 500 liters, less than 1000 liters, or more than 1000 liters of the probiotic starting culture may be added to one or more primary tanks. In some embodiments, at least 625.2 liters of the probiotic starting culture may be added to one or more primary tanks. Relatedly, and without limitation, less than 10 gallons, less than 500 gallons, less than 1000 gallons, or more than 1000 gallons of the probiotic starting culture may be added to one or more primary tanks. In some embodiments, at least 165.2 gallons of the probiotic starting culture may be added to one or more primary tanks. Moreover, and without limitation, less than 10 kg, less than 500 kg, less than 1000 kg, or more than 1000 kg of the probiotic starting culture may be added to one or more primary tanks. In some embodiments, at least 625.2 kg of the probiotic starting culture may be added to one or more primary tanks. The probiotic starting culture may comprise less than 10%, less than 25%, less than 50%, less than 75%, less than 95%, or less than 99.9% of the total in-tank solution. In some embodiments, the probiotic starting culture comprises 60% of the total in-tank solution.

Next, one or more volumes of one or more additional elements may be added to the probiotic starting culture in one or more primary fermentation tanks. The additional element(s) may be added to the probiotic starting culture at an amount less than 10 liters, less than 500 liters, less than 1000 liters, or more than 1000 liters in the one or more primary tanks. In some embodiments, at least 104.2 liters of the additional element(s) may be added to the probiotic starting culture in the one or more primary tanks. Relatedly, and without limitation, less than 10 gallons, less than 500 gallons, less than 1000 gallons, or more than 1000 gallons of the additional element(s) may be added to the probiotic starting culture in the one or more primary tanks. In some embodiments, at least 27.5 gallons of the additional element(s) may be added to the probiotic starting culture in the one or more primary tanks. Moreover, and without limitation, less than 10 kg, less than 500 kg, less than 1000 kg, or more than 1000 kg of the additional element(s) may be added to the probiotic starting culture in the one or more primary tanks. In some embodiments, at least 104.2 kg of the additional element(s) may be added to the probiotic starting culture in the one or more primary tanks. The additional element(s) may comprise less than 10%, less than 25%, less than 50%, less than 75%, less than 95%, or less than 99.9% of the total in-tank solution. In some embodiments, the additional element(s) comprises 10% of the total in-tank solution. The product of the probiotic starting culture and the additional element(s) may comprise one or more fermented product(s).

In some embodiments, the probiotic starting culture, the probiotic starting culture plus additional element(s), and/or the fermented product may be agitated. In some embodiments, the agitation may be constant. In some embodiments, the agitation may be constant and may last for one minute, less than thirty minutes, less than 1 hour, less than 12 hours, less than 24 hours, or more than 24 hours. In some embodiments, the fermented product may be constantly agitated for at least 1 hour.

Following fermentation, the fermented product may be extracted or decanted.

Following agitation, in some embodiments, the biodegradable cleaning composition may be extracted or decanted.

The extraction and/or decanting step may comprise passing the fermented product through a 30-micron filter, feeding the fermented product into a centrifuge at a feed rate of 150 cc/minute, then centrifuging the fermented product at 22,000-23,000 revolutions per minute.

In some embodiments, the fermented product may be concentrated. In some embodiments, the fermented product may be concentrated before being extracted. In some embodiments, the fermented product may be concentrated after being extracted.

In some embodiments, the step of concentrating the fermented product or the biodegradable cleaning composition product may comprise decanting the fermented product or biodegradable cleaning composition. The decanting step may utilize any techniques or decanting equipment known in the art. In some embodiments, the decanting step may be performed until a suitable product is formed and collected. At any step in the method, including but not limited to one or more fermentation steps, some of all of the probiotic starting culture, the fermented product, or any form of the biodegradable cleaning composition, as applicable, may be held within a primary fermentation tank, a horizontal fermentation tank, a vertical fermentation tank, or any combination of tanks.

In other embodiments, the step of concentrating the fermented product or biodegradable cleaning composition may comprise passing the fermented product through a gravity filtration apparatus, evaporating the fermented product or biofertilizer via an evaporator, evaporating the fermented product or biofertilizer via passive evaporation, or concentration by pervaporative separation of the fermented product or biofertilizer via a pervaporation system.

At any point or step of the method, any mixture, composition, or product of the present disclosure may undergo a fermentation. For example, the probiotic starting culture (previously or concurrently with its presence in one or more fermentation tanks), the first modified composition, the second modified composition, or the fermented product, as applicable, may undergo one or more additional or alternate fermentations. Each of the one or more fermentations may comprise any amount of time known or anticipated to be useful, such as but not limited to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more days. Preferably, the composition is fermented for at least about 15 to about 23 days. More preferably, the probiotic starting culture, the first modified composition, the second modified composition, or the fermented product, as applicable, may be fermented for at least 21 days. More preferably, the probiotic starting culture, the first modified composition, the second modified composition, or the fermented product, as applicable, may be fermented for at least 15 days.

The decanted product of the fermented product, in some embodiments, may be the biodegradable cleaning composition, which generally is present in liquid form.

In some embodiments, the method further comprises the step of removing water from the biodegradable cleaning composition to form a solid biodegradable cleaning composition. In some embodiments, the solid biodegradable cleaning composition may comprise ≤10% water (w/w).

EXAMPLES

Test Method: ASTM D4488-A5 Particulate and Oily Soil/Vinyl Tiles Test Method

Procedure: A synthetic particulate soil is prepared according to ASTM D4488-95 (2001) A5. An amount of 50 mg of this particulate soil is placed on a white vinyl tile over an area of 2×4 in. as defined with a template. The soil mound is covered with a piece of paper towel and 5 drops of an oil blend is applied to the soil. The soil is rubbed over the 2×4 in area in a circular motion. The soiled tile is allowed to air dry for 24 hours. Reflectance readings are recorded from the clean tile, the soiled tile, and the cleaned tile. Cleaning is performed by adding 50 ml of the cleaning solution to a sponge. The soiled area is covered with 20 ml of the cleaning solution for 60 seconds and then scrubbed with the sponge for 10 cycles with a gardner Straight-line Washability Apparatus.

Calculation of Cleaning Efficiency:Cleaning efficiency (%)=(R1−R2)/(R3−R2)

Where: R1=reflectance of soiled tile after cleaningR2=reflectance of soiled tile before cleaningR3=reflectance of tile before soiling

As may be seen inFIG.3, the below images illustrate some aspects of Experiment 1:

These results improved on previous results, here reproduced per the table below:

Summary of Cleaning Efficiency Per ASTM 4499-95 (A5)

As can be seen inFIGS.1-18, untargeted metabolomics analysis was conducted on or around Sep. 1, 2020 on two samples of the biogradable cleaning composition. The results indicated:

On or around Sep. 1, 2020, experiments were run on at least one biodegradable cleaning composition described herein per the following testing methodologies:

Lactic Acid Bacteria testing: Serial dilutions of the samples are performed using peptone water and the resulting dilutions are cultured on the Lactobacilli MRS agar media by pour plate method. The media plates are then incubated anaerobically at 37° C. for 3-5 days. The counts from the plate within the range of 25-250 colonies are counted and reported as CFU/mL.

Bacillustesting: Serial dilutions of the samples are performed using peptone water and the resulting dilutions are cultured on the Tryptic soy agar media by spread plate method. The media plates are incubated aerobically at 35° C. upto 3 days. The counts from the plate within the range of 25-250 colonies are counted and reported as CFU/mL

Yeast and Mold testing: The samples are cultured on Potato dextrose agar media and the plates are incubated in the incubator at 32° C. for 3 days for yeast counts and 5 days for the mold testing.

Coliform/E colitesting: The samples are cultured on the freshly made Violet Red Bile Agar media by pour plated method. The plates are incubated in the incubator for 18-24 hrs. at 35-37° C. 3M™ Petrifilm™E. coli/Coliform Count Plate can be used as an alternative to Violet red bile agar and same incubation procedures are followed. Red or blue colonies with associated gas colonies are counted as coliform colonies.

Pathogen testing/challenge: The time-kill test involves inoculating the sample with a known concentration of pathogenic microorganisms and sampling them immediately after inoculation and 1 hour, 3 hours, 7 hours, 24 hours, and 48 hours after inoculation. The purpose of this test is to determine how rapidly the sample can eradicate pathogenic microorganisms.

The challenge test involves inoculating a sample with a known concentration of the microorganisms to test, such asCandida albicans, Staphylococcus aureus, Aspergillus brasieliensis, etc. The sample is tested for the presence of the microorganisms immediately after inoculation, as well as 7, 14, and 28 days after inoculation. The concentration is determined at each timepoint and compared to the initial recovery to determine the percent reduction. The purpose of this test is to determine whether the product is sufficiently capable of killing any undesirable microorganisms that may be present.

% TA testing: % Titratable Acidity is the measure of the amount of acid present in a substance. It is determined by the amount of strong base required to neutralize the acid. Lactic acid is formed as a result of fermentation process as secondary metabolite and the % of lactic acid is determined by amount of 1N sodium hydroxide used to neutralize the acid. % lactic acid=((mL NaOH×N NaOH×milliequivalent weight of lactic acid*)×100)/(volume of Product Sample in mL)

Surface Tension testing: Surface tension is measured are made using the Force tensiometer by Du Noüy ring method. The values are reported as N/m (Newton/meter) or dyn/cm (dyne per centimeter)

Spore Germination testing: This test is done to demonstrateBacillusspore germination on surface for competitive exclusion. The sample is inoculated on 2 sets of 10 cm×10 cm surface and they are incubated at ambient temperature. One set of hard surface is washed with PBS buffer and the eluted samples in divided into two parts. One part of sample is plated at T0 and another sample incubated at 72° C. for 30 min and then plated on TSA media and incubated up to 24-72 hrs. The microbial colony counts are recorded. At 24 hrs. another set of prepped hard surface is tested and processed as T24 samples.

Surface Cleaning of Pathogens testing: This test is done to demonstrate the cleaning product's ability to remove undesirable microorganisms from surface and to inhibit re-contamination. A cell suspension containingS. aureus, E. coli, Salmonellasp. andP. aeruginosa, A. fumigatusis seeded on a sterile 10 cm×10 cm surface (100 cm2) at ambient temperature. The surface is treated with the cleaning product and wiped off. The resulting surface is washed off with PBST buffer and the samples are plated on MacConkey media forE. coli, XLD media forSalmonellasp. Cetrimide media forP. aeruginosa, Baird-Parker media forS. aureus, and PDA forA. fumigatus. at T0, and the same steps are repeated at T7, T24, and T48. The inoculated plates are then incubated at 35±2 0 C for 24-72 hrs. and at 25±2 0 C for 72-140 hrs. for fungal samples. The resulting colonies counts are recorded as CFU/mL

Surface

Testing

Spore Germination

Spore Germination

Stability of Z1

Stability of Z-2

Embodiments

In one or more embodiments, the biodegradable cleaning composition may comprise: a probiotic starting culture comprising at least one species of microbe selected from the group consisting of at least one species of lactic acid bacteria and at least one species ofBacillus; at least one carbon source, and dechlorinated water; and at least one added element selected from the group consisting of at least one essential oil, carbon source, biodetergent, organic acid, preservative, diol, polyol, polymyxin, polysaccharide-lipid complex, fatty acid, terpene, and preservative.

In one or more embodiments the at least one species of lactic acid bacteria may be selected from the group consisting of a species ofCarnobacteriumsp.,Enterococcussp.,Lactobacillussp.,Lactococcussp.,Leuconostocsp.,Oenococcussp.Streptococcussp.,Tetragenococcussp.,Vagococcussp., andWeissellasp.

In one or more embodiments the at least one species of lactic acid bacteria may be selected from the group consisting of a species ofCarnobacteriumsp.,Enterococcussp.,Lactobacillussp.,Lactococcussp., andStreptococcussp.

In one or more embodiments the at least one species ofBacillusmay be selected from the group consisting ofBacillus licheniformis, Bacillus coagulans, andBacillus subtilis.

In one or more embodiments the at least one species ofBacillusmay be selected from the group consisting ofBacillus coagulansandBacillus subtilis.

In one or more embodiments the at least one carbon source may be selected from the group consisting of rum, molasses, glucose, starch, cellulose, fructose, sucrose, arabinose, mannose, and maltose.

In one or more embodiments the at least one added element may comprise at least one biodetergent selected from the group consisting of surfactin, iturin, fengycin, arthrofactin, emulsan.

In one or more embodiments the at least one added element may be a biodetergent produced by at least one species of microbe selected from the group consisting ofBacillussp.,Bacillus atrophaeus, Bacillus licheniformis, Bacillus subtilis, Bacillus siamensis, Bacillus thuringiensis, Bacillus pumilus, Bacillus cereus, Torulopsissp.,Pseudomonassp.,Rhodococcussp.,Candidasp.,Candida bogoriensis, Acinetobactersp.,Corynebacterium lepus, Candida petrophilum, Candida tropicalis, Corynebacterium hydrocarboclastus, Flavobacteriumsp.,Achromobactersp.,Arthrobactersp.,Micrococcussp., andPaenibacillussp.

In one or more embodiments the at least one added element may comprise at least one diol selected from the group consisting of 1,2-propanediol (propylene glycol), 2,2-propanediol, 1,1-propanediol, 1,3-propanediol, butane diol(s), 1,4-butanediol, ethylene glycol, geminal diols, vicinal diols, 1,3-diols, 1,4-diols, 1,5-diols, longer than 1,5-diols, 5-(1-Hydroxy-1-methylethyl)-2-methyl-2-cyclohexene-1,4-diol, and aliphatic diols.

In one or more embodiments the at least one added element may comprise at least one polymyxin may be selected from the group consisting of polymyxin A, polymyxin B, polymyxin B1, polymyxin B nonapeptide, polymyxin C, polymyxin D, and polymyxin E (colistin), and other cationic cyclic decapeptides.

In one or more embodiments the at least one added element may comprise at least one polysaccharide-lipid complex selected from the group consisting of a lipopolysaccharide, lipooligosaccharide, or endotoxins produced by a microbe of the group comprising but not limited toE. coliO26,E. coliEH-100,E. coli0111,E. coli055,E. coli0127,E. coli0128,E. coliF-583,E. coliJ5,E. coliK-235,Pseudomonas aeroginosa10,Salmonella, Salmonella enteritidis, Salmonella minnesota, Salmonella minnesotastrain Re595,Salmonella typhimurium, Salmonella typhimuriumstrain SL1181,Salmonella typhimuriumstrain TV119,Salmonella typhosa, andSerratia marcescens.

In one or more embodiments the at least one preservative may be present in a ratio of from about 0.1-0.3 wt/wt %.

In one or more embodiments the biodegradable cleaning composition may have a surface tension between about 20 dyne/cm and about 38 dyne/cm.

In one or more embodiments the biodegradable cleaning composition may comprise about 80 to about 85 wt/wt % dechlorinated water.

In one or more embodiments the biodegradable cleaning composition may comprise about 90 to about 95 wt/wt % dechlorinated water.

An embodiment comprising a kit comprising a textile treated with an application of the biodegradable cleaning composition of any one of claims1-16, wherein the application may be performed according to a ratio, and wherein said textile may be contained within an openable container.

An embodiment comprising a kit wherein the ratio may be from about 5 ml per textile sheet to about 15 ml per textile sheet.

An embodiment comprising a kit wherein the textile may be selected from the group consisting of lyocell, rayon, cotton polyester, and polypropylene.

An embodiment comprising a method of manufacturing a biodegradable cleaning composition comprising the steps of:obtaining a probiotic starting culture comprising at least one species of microbe selected from the group consisting of at least one species of lactic acid bacteria and at least oneBacillusspecies; at least one organic acid; at least one carbon source; and dechlorinated water;beginning a fermentation period, wherein the probiotic starting culture may be fermented in at least one fermentation tank for at least one fermentation period;adding at least one additional element selected from the group consisting of at least one essential oil, carbon source, biodetergent, organic acid, preservative, diol, polyol, polymyxin, polysaccharide-lipid complex, fatty acid, terpene, and preservative.allowing the probiotic starting culture and the at least one additional element to ferment into a fermented product;ending the fermentation period;decanting the fermented product; andextracting the biodegradable cleaning composition.

An embodiment comprising a method, wherein a decanting step may be performed using a solid-liquid separation centrifuge operating at 22,000-23,000 revolutions per minute.

An embodiment comprising a method, wherein a decanting step further comprises passing the fermented product into the centrifuge at a material feed rate of 150 cc/minute.

An embodiment comprising a method, wherein the decanting step further comprises passing the fermented product through a 30-micron filter one or more times.

An embodiment of the biodegradable cleaning composition produced by an embodiment comprising a method, including any modifications thereof, such as but not limited to modifications of said method comprising modifications to a decanting step.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.1.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.2.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.3.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.4.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.5.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.6.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.7.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.8.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.9.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.10.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.11.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.12.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.13.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.14.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.15.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.16.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.17.

One or more embodiments of the biodegradable cleaning composition may be comprised according toFIG.18.

In one or more embodiments, a biodegradable cleaning composition may comprise (i) purified water; (ii) a co-culture of lactic acid bacteria comprising two or more species of lactic acid bacteria; (iii) lactic acid; and (iv) at least one organic essential oil.

In one or more embodiments, a biodegradable cleaning composition may comprise (i) purified water; (ii) a co-culture comprising one or more species of lactic acid bacteria and one or more species ofBacillus; (iii) lactic acid; and (iv) at least one organic essential oil.

In one or more embodiments, the at least one organic essential oil may comprise geranium.

In one or more embodiments, the at least one organic essential oil may comprise lemongrass oil.

In one or more embodiments, the at least one organic essential oil may comprise lavender oil.

In one or more embodiments, the biodegradable cleaning composition may be formulated for use as an all-purpose cleaning solution.

In one or more embodiments, the biodegradable cleaning composition may be formulated for use as an air freshening solution.

In one or more embodiments, the biodegradable cleaning composition may be formulated for use as an air-and-fabric freshening solution.

In one or more embodiments, the biodegradable cleaning composition may be formulated for use as a floor cleaning solution.

In one or more embodiments, the biodegradable cleaning composition may be formulated for use as a floor cleaning concentrate solution.

In one or more embodiments, a biodegradable cleaning composition may comprise a co-culture comprising at least one species of microbe; at least one organic acid; at least one carbon source; and dechlorinated water.

In one or more embodiments, the biodegradable cleaning composition may be decanted prior to administration, wherein said decanting is performed using a solid-liquid separation centrifuge.

In one or more embodiments, the at least one species of microbe may be selected from the group consisting of one or more species of lactic acid bacteria and one or more species ofBacillus.

In one or more embodiments, the at least one organic acid may comprise at least one organic acid.

In one or more embodiments, the at least one organic acid may comprise at least one fatty acid.

In one or more embodiments, the at least one fatty acid may comprise an unsaturated fatty acid.

In one or more embodiments, the at least one fatty acid may comprise a saturated fatty acid.

In one or more embodiments, the biodegradable cleaning composition may further comprise at least one polyol.

In one or more embodiments, the biodegradable cleaning composition may further comprise at least one diol.

In one or more embodiments, the biodegradable cleaning composition may further comprise at least one polyethylene glycol.

In one or more embodiments, the biodegradable cleaning composition may further comprise at least one polysaccharide-lipid complex.

In one or more embodiments, the biodegradable cleaning composition may further comprise at least one monoterpene.

In one or more embodiments, the biodegradable cleaning composition may further comprise at least one sesquiterpene.

In one or more embodiments, the biodegradable cleaning composition may further comprise at least one essential oil.

In one or more embodiments, the biodegradable cleaning composition may further comprise at least one polymyxin;

In one or more embodiments, the biodegradable cleaning composition may further comprise at least one biodetergent.

In one or more embodiments, a biodegradable cleaning composition may comprise at least one species of microbe selected from the group consisting of lactic acid bacteria andBacillus; at least one carboxylic acid; at least one carbon source; dechlorinated water; and one or more added element, wherein the added element is selected from the group comprising at least one polyol, at least one diol, at least one polyethylene glycol, at least one polysaccharide-lipid complex, at least one monoterpene, at least one sesquiterpene, at least one essential oil, at least one polymyxin, and at least one biodetergent.

In one or more embodiments, a biodegradable cleaning composition may comprise at least one species of microbe selected from the group consisting of lactic acid bacteria andBacillus; at least one carboxylic acid; at least one carbon source; dechlorinated water; at least one polyol, at least one diol, at least one polyethylene glycol, at least one polysaccharide-lipid complex, at least one monoterpene, at least one sesquiterpene, at least one essential oil, at least one polymyxin, and at least one biodetergent.

In one or more embodiments, a biodegradable cleaning composition may comprise

In one or more embodiments, the at least one species of lactic acid bacteria is selected from the group consisting of a species ofCarnobacteriumsp.Enterococcussp.Lactobacillussp.Lactococcussp.Leuconostocsp.Oenococcussp.Streptococcussp.Tetragenococcussp.Vagococcussp. andWeissellasp.

In one or more embodiments, the at least one species ofBacillusis selected from the group consisting ofBacillus cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus licheniformis, Bacillus pumilus, Bacillus mycoides, Bacillus coagulans, Bacillus megaterium, andBacillus subtilis.

In some embodiments, the at least one unsaturated fatty acid may be present at a molar concentration of 0.1M to 1M.

In some embodiments, the at least one saturated fatty acid may be present at a molar concentration of 0.1M to 1M.

In one or more embodiments, the at least one diol is selected from the group consisting of 1,2-propanediol (propylene glycol), 2,2-propanediol, 1,1-propanediol, 1,3-propanediol, butane diol(s), 1,4-butanediol, ethylene glycol, geminal diols, vicinal diols, 1,3-diols, 1,4-diols, 1,5-diols, longer than 1,5-diols, 5-(1-Hydroxy-1-methylethyl)-2-methyl-2-cyclohexene-1,4-diol, and aliphatic diols.

In some embodiments, the biodegradable cleaning composition may further comprise at least one diterpenes, sesterterpenes triterpenes, sesquarterpenes, tetraterpenes, polyterpenes, norisoprenoids or ursane-type triterpenes.

In some embodiments, the at least one polymyxin may be selected from the group consisting of polymyxin A, polymyxin B, polymyxin B1, polymyxin B nonapeptide, polymyxin C, polymyxin D, and polymyxin E (colistin), and other cationic cyclic decapeptides.

In some embodiments, the at least one added element may comprise a hydrocarbon, such as but not limited to cyclohexane, cyclohexene, 3-acetoxy-4-(1-hydroxy-1-methylethyl)-1-methyl, cycloehexane, an aldehyde such as but not limited to 2-furaldehyde (furfural), a ketone such as but not limited to sulcatone, nonanone, or 4-nonanone, tagalose, D-(−)-tagalose, D-(+)-tagalose, mannopyranose, b-D-(+)-mannopyranose, b-D-(−)-mannopyranose, sulctone, 2,3-dehydro-1,8-cineole, a-phellandren-8-ol, 2,6-octadien-1-ol, 3,7-dimethyl-acetate, caryophyllene, g-cadinene, cadina-1(10),4-diene, carophyllene oxide, [(2-ethyl-5-methylfuran-3,4-diyl)bis(oxy)]bis(trimethylsilane), a polymer such as siloxane or pentasiloxane, or a gas such as silane or [(2-Ethyl-5-methylfuran-3,4-diyl)bis(oxy)]bis(trimethylsilane).

In some embodiments, the at least one biodetergent may comprise one or more of surfactin, iturin, fengycin, arthrofactin, emulsan, or another biodetergent known to be applicable in leather tanning processes.

In some embodiments, the at least one biodetergent may comprise one or more biodetergent produced byBacillussp.Bacillus atrophaeus, Bacillus licheniformis, Bacillus subtilis, Bacillus siamensis, Bacillus thuringiensis, Bacillus pumilus, Bacillus cereus, Torulopsissp.Pseudomonassp.Rhodococcussp.Candidasp.Candida bogoriensis, Acinetobactersp.Corynebacterium lepus, Candida petrophilum, Candida tropicalis, Corynebacterium hydrocarboclastus, Flavobacteriumsp.Achromobactersp.Arthrobactersp.Micrococcussp.Paenibacillussp. or any other microbial species named herein or known to produce biodetergents.

In some embodiments, the at least one biodetergent is surfactin.

In one or more embodiments, the biodegradable cleaning composition may be decanted prior to extraction from a fermentation tank.

In one or more embodiments, the biodegradable cleaning composition may be decanted using a solid-liquid separation centrifuge prior to extraction from a fermentation tank.

In one or more embodiments, the biodegradable cleaning composition has a surface tension between about 20 dyne/cm and about 38 dyne/cm.

In one or more embodiments, a biodegradable cleaning composition may comprise:a. a co-culture comprising at least one species of microbe selected from the group consisting of at least one species of lactic acid bacteria, at least one species ofBacillus, and at least one yeast;b. at least one carboxylic acid;c. at least one carbon source;d. dechlorinated water; ande. at least one added element, wherein the added element is selected from the group comprising at least one polyol, at least one diol, at least one polyethylene glycol, at least one polysaccharide-lipid complex, at least one monoterpene, at least one sesquiterpene, at least one essential oil, at least one polymyxin, and at least one biodetergent.

In one or more embodiments, the at least one species of lactic acid bacteria is selected from the group consisting of a species ofCarnobacteriumsp.Enterococcussp.Lactobacillussp.Lactococcussp.Leuconostocsp.Oenococcussp.Streptococcussp.Tetragenococcussp.Vagococcussp. andWeissellasp.

In one or more embodiments, the at least one species ofBacillusis selected from the group consisting ofBacillus cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus licheniformis, Bacillus pumilus, Bacillus mycoides, Bacillus coagulans, Bacillus megaterium, andBacillus subtilis.

In one or more embodiments, the at least one unsaturated fatty acid is present at a molar concentration of 0.1M to 1M.

In one or more embodiments, the at least one saturated fatty acid is present at a molar concentration of 0.1M to 1M.

In one or more embodiments, the at least one diol is selected from the group consisting of 1,2-propanediol (propylene glycol), 2,2-propanediol, 1,1-propanediol, 1,3-propanediol, butane diol(s), 1,4-butanediol, ethylene glycol, geminal diols, vicinal diols, 1,3-diols, 1,4-diols, 1,5-diols, longer than 1,5-diols, 5-(1-Hydroxy-1-methylethyl)-2-methyl-2-cyclohexene-1,4-diol, and aliphatic diols.

In one or more embodiments, the at least one polysaccharide-lipid complex is selected from the group consisting of a lipopolysaccharide, lipooligosaccharide, or endotoxins produced by a microbe of the group comprising but not limited toE. coliO26,E. coliEH-100,E. coli0111,E. coli055,E. coli0127,E. coli0128,E. coliF-583,E. coliJ5,E. coliK-235,Pseudomonas aeroginosa10,Salmonella, Salmonella enteritidis, Salmonella minnesota, Salmonella minnesotastrain Re595,Salmonella typhimurium, Salmonella typhimuriumstrain SL1181,Salmonella typhimuriumstrain TV119,Salmonella typhosa, andSerratia marcescens.

In one or more embodiments, the at least one polymyxin is selected from the group consisting of polymyxin A, polymyxin B, polymyxin B1, polymyxin B nonapeptide, polymyxin C, polymyxin D, and polymyxin E (colistin), and other cationic cyclic decapeptides.

In one or more embodiments, the at least one biodetergent is selected from the group consisting of surfactin, iturin, fengycin, arthrofactin, emulsan.

In one or more embodiments, the at least one biodetergent is a biodetergent produced by at least one species of microbe selected from the group consisting ofBacillussp.Bacillus atrophaeus, Bacillus licheniformis, Bacillus subtilis, Bacillus siamensis, Bacillus thuringiensis, Bacillus pumilus, Bacillus cereus, Torulopsissp.Pseudomonassp.Rhodococcussp.Candidasp.Candida bogoriensis, Acinetobactersp.Corynebacterium lepus, Candida petrophilum, Candida tropicalis, Corynebacterium hydrocarboclastus, Flavobacteriumsp.Achromobactersp.Arthrobactersp.Micrococcussp. andPaenibacillussp.

In one or more embodiments, the biodegradable cleaning composition is decanted prior to extraction from a fermentation tank, and wherein said decanting is performed using a solid-liquid separation centrifuge.

In one or more embodiments, the biodegradable cleaning composition has a surface tension between about 20 dyne/cm and about 38 dyne/cm.

In one or more embodiments, a method of manufacturing one or more biodegradable cleaning compositions may comprise the steps ofa. obtaining a probiotic starting culture comprising at least one species of microbe selected from the group consisting of at least one species of lactic acid bacteria and at least oneBacillusspecies; at least one organic acid; at least one carbon source; and dechlorinated water;b. beginning a fermentation period, wherein the probiotic starting culture is fermented in at least one fermentation tank for at least one fermentation period;c. adding at least one additional element to the probiotic starting culture, wherein the at least one additional element comprises one or more of at least one essential oil; at least one monoterpene; at least one sesquiterpene; at least one essential oil, at least one carboxylic acid; at least one organic acid; at least one fatty acid; at least one polyol; at least one diol; at least one polyethylene glycol; at least one polysaccharide-lipid complex; at least one polymyxin; and at least one biodetergent;d. allowing the probiotic starting culture and the at least one additional element to ferment into a fermented product;e. ending the fermentation period; andf. decanting the fermented product into a biodegradable cleaning composition.

In one or more embodiments, the fermentation period may last at least one day.

In one or more embodiments, the fermentation period may last 14 days.

In one or more embodiments, the fermentation period may last 14 days or more.

In one or more embodiments, the at least one additional element may comprise one or more stabilizer, microbial inoculant, antioxidant, or preservative.

In one or more embodiments, the decanting step may be performed using a solid-liquid separation centrifuge.

In one or more embodiments, the decanting step may be performed using a centrifuge operating at 22,000-23,000 revolutions per minute.

In one or more embodiments, the decanting step may further comprise passing the fermented product into the centrifuge at a material feed rate of 150 cc/minute.

In one or more embodiments, the step of decanting the fermented product may further comprise passing the fermented product through a micron filter one or more times.

In one or more embodiments, the micron filter may be a 30 micron filter.

In one or more embodiments, a biodegradable cleaning composition may be produced by any process described herein.

In one or more embodiments, a biodegradable cleaning composition may be produced by any method described herein.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

The term “about” means within the inclusive range of ±15% of the numerical value it modifies.

As used herein, “administering” is used in its broadest sense to mean contacting a subject, surface, liquid, or environment with a composition of the invention.

The term “culture” refers to a culture of microorganisms that includes at least two microorganisms of the present disclosure, described herein. The term “brew” may be used in place of “culture”. The terms “culture and “co-culture” may be used interchangeably.

The term “detectable” as used herein means at or above the limit of detection of a colony-forming unit of particular bacteria, yeast, or other microbes in a microorganism consortium, or at or above the limit of detection of a element or compound, either free or in solution. Any element, compound, or species detected by any known method constitutes “detected”.

Methods for determining the presence of (detecting) a particular species or strain of microbe include culture techniques and non-culture techniques. Culture techniques include the use of selective agar media and determination of colony morphology, such as e.g., LAMVAB and Rogosa agar for Lactobacilli ssp., liver-cysteine-lactose and raffinose forBifidobacteriumssp., heterotrophic plate counting, and the like. Non-culture techniques include e.g., flow cytometry, direct epifluorescent counting, PCR and other DNA-based methods, such as quantitative PCR, and metabolic/chemical methods, such as propidium monoazide PCR or ethidium monoazide PCR. Methods of detection useful in the practice of this invention are described in Catherine Davis, “Enumeration of biofertilizer strains: Review of culture-dependent and alternative techniques to quantify viable bacteria,” Journal of Microbiological Methods, Volume 103, 2014, pp. 9-17; Jackson and Bird, “Comparison of two selective media for the detection and enumeration of Lactobacilli in human faeces,” Journal of Microbiological Methods 51 (2002) 313-321; Lu et al., “Fine Structure of Tibetan Kefir Grains and Their Yeast Distribution, Diversity, and Shift,” PLoS One. 2014; 9(6): e101387; Rachbid et al., “Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis,” Food Microbiology, Volume 31, Issue 2, September 2012, Pages 215-221; Furet et al., “Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR,” International Journal of Food Microbiology, Volume 97, Issue 2, 15 Dec. 2004, Pages 197-207; and Garcia-Cayuel et al., “Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR,” International Dairy Journal, Volume 19, Issues 6-7, June-July 2009, Pages 405-409.

The phrase “fermentation medium” may refer to a mixture including at least one microorganism, expression products of the microorganism(s), substances produced by the microorganisms, extracts of the microorganisms, and a culture medium or other elements of a culture medium. In some embodiments, the expression product or substance produced by a microorganism may comprise a gas or organic compound product of the fermentation of a carbohydrate, such as an organic acid like acetic acid, citric acid, gluconic acid, lactic acid, propionic acid, pyruvic acid, or succinic acid, a gas like carbon dioxide, or an alcohol like ethanol. In a preferred embodiment, the expression product or substance produced by a microorganism is lactic acid, ethanol, or a combination of lactic acid and ethanol.

“Fermentation product” refers to both or either organic chemical products of a fermentation reaction (e.g., carbon dioxide, ethanol, lactic acid, propionic acid), and/or the fermented carbon source (e.g., fermented sugar cane molasse, fermented rice bran).

The term “finished product” refers to a mixture including a fermentation product. The finished product may include additional additives.

The term “monoculture” refers to a culture having a single species of microorganism.

The phrase “non-pathogenic gram-positive Bacilli bacteria” refers to those bacteria that belong to the Bacilli taxonomic class of bacteria that contains two orders, Bacillales and Lactobacillales. The term “gram-positive” is used herein to distinguish the subject Bacilli from the group of gram-negative rod-shaped bacteria that are sometimes referred to as bacilli, including for exampleEscherichia coliand other coliform bacteria. The term “non-pathogenic” is used to refer to those Bacilli that do not cause disease or harm to a plant or animal, i.e., excluding pathogenic bacteria. For example, certain species ofBacillus(e.g., anthraces),Listeria, Staphylococcus, andStreptococcusare pathogenic and are excluded. Non-limiting examples of non-pathogenic gram-positive Bacilli bacteria includeBifidobacteriumspp.,Lactobacillusspp.,Lactococcusspp.,Carnobacteriumspp.,Streptococcusspp., andBacillusspp.

The term “microorganism consortium” refers to a composition that contains a live beneficial or useful microorganism alone, in combination with another microorganism, and/or combined with other ingredients such as e.g., energy sources, pre-biotics, stabilizers, and the like, in culture media. Beneficial microorganisms are generally known in the art and include such bacteria as lactic acid fermenting (obligative and facultative) bacteria, phototrophic bacteria, and non-pathogenic bacilli, as well as fermenting yeast such as the Saccharomycetaceae. Microorganism consortiums are generally known to be useful in ameliorating gut flora, remediating wastewater, treating microbial imbalances in animals and plants, protecting animals, plants, and soil from harmful microbes, and improving food animal and food plant production and yield. Useful biofertilizer microorganisms can be found listed for example in Biofertilizer Bacteria: Fundamentals, Therapy, and Technological Aspects, edited by J. Paulo Sousa e Silva, Ana Cristina Freitas, CRC Press, Apr. 2, 2014; and The European Union Register of Feed Additives pursuant to Regulation (EC) No 1831/2003, Annex I: List of additives, available at https://ec.europa.eu/food/sites/food/files/safety/docs/animal-feed-eu-regcomm_register_feed_additives_1831-03.pdf.

The term “probiotic starting culture” refers to a microorganism consortium prior to combination with other ingredients.

The term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a composition. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of pharmaceutical compositions are known in the art and may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

As various changes could be made in the above compositions and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the Examples given below, shall be interpreted as illustrative and not in a limiting sense.