Microbial-enhancing composition containing a surfactant blend that overcomes antagonistic surfactant incompatibility.

Abstract

A composition is disclosed that can overcome the adverse effects that certain surfactants have on the viability, growth, and biological activity of beneficial microorganisms. The composition comprises at least one anionic surfactant and at least one amphoteric surfactant or glyceryl monoester and / or glyceryl diester, each of which, individually, adversely affects the viability, growth, and / or biological activity of beneficial microorganisms, but when used in combination, overcomes the adverse effects that each surfactant has on beneficial microorganisms. The composition further comprises beneficial microorganisms and, optionally, at least one nonionic surfactant that adversely affects the viability, growth, and / or biological activity of beneficial microorganisms. A method is also disclosed for overcoming the adverse effects that certain surfactants have on beneficial microorganisms by combining a selected anionic surfactant with an amphoteric surfactant or glyceryl monoester and / or glyceryl diester in specific amounts and ratios with beneficial microorganisms.

Description

[Technical Field]

[0001] This technology relates to a composition comprising at least two surfactants that, individually, adversely affect the viability, growth, and / or biological activity of beneficial microorganisms, but which, when combined, overcome the adverse effects of surfactants and thereby promote the viability, growth, and biological activity of beneficial microorganisms. This technology also relates to a method for overcoming the adverse effects that certain surfactants have on beneficial microorganisms by combining those surfactants in specific amounts and ratios that overcome the adverse effects on the microorganisms. [Background technology]

[0002] A recent trend is to formulate products with ingredients based on renewable raw materials rather than fossil fuels. Such ingredients are considered "green" or "natural" because they originate from renewable and / or sustainable sources. As a result, they are more environmentally friendly than ingredients derived from fossil fuels. Ingredients with a high Bio-Renewable Carbon Index (BCI) of over 80 indicate that the ingredient contains carbon primarily derived from plant, animal, or marine organism-based sources.

[0003] To enable improved results, there has been a recent trend to formulate products using whole cells of beneficial microorganisms, such as various Bacillus species. For example, bacterial endospores (commonly referred to as spores) may be added to cleaning formulations to obtain long-lasting cleaning effects. Bacterial spores can also be used in agricultural applications to enhance plant health and vitality, and in bioremediation compositions to further accelerate the biodegradation of organic contaminants. In spore form, beneficial microorganisms do not provide any specific benefits to a composition. To obtain performance benefits, spores must germinate, grow, and exhibit biological activity when needed. If environmental conditions such as pH, ionic strength, and nutrients for food (such as proteins and oils) are favorable, beneficial microbial spores can germinate and transform into vegetative cells, at which point the microorganisms produce enzymes and metabolites, digest organic matter (e.g., embedded dirt), solubilize inorganic plant nutrients, promote a healthy microbiome, and eliminate undesirable and pathogenic microorganisms.

[0004] A crucial issue for germination, growth, and biological activity is the compatibility between beneficial microorganisms and other components in the formulation. Currently, many surfactants used in, for example, cleaning and agricultural compositions, have been found to create unfavorable environments for microorganisms. Surfactants that can adversely affect the viability of beneficial microorganisms include many common anionic surfactants such as alkyl sulfates and alkyl ether sulfates, as well as most amphoteric surfactants. Another surfactant that adversely affects beneficial microorganisms is rhamnolipid, a surfactant glycolipid produced by various bacterial species. Adding beneficial microorganisms to compositions containing these surfactants does not yield the desired benefits because the beneficial microorganisms are unable to germinate, or, after germination, cannot grow, reproduce, or survive.

[0005] Another important consideration for compositions containing beneficial microorganisms is their ability to maintain a stable suspension of microorganisms during storage and use. Microorganisms that settle at the bottom of the container may not be properly dispersed by shaking or stirring, resulting in uneven or inconsistent application of the microorganisms.

[0006] Therefore, there is a need for a composition containing beneficial microorganisms that can maintain their viability during storage and allow beneficial microorganisms to germinate and proliferate when used. Furthermore, there is a need for a composition that is storage stable and can maintain a suspended state of microorganisms without aggregation or precipitation.

[0007] The applicants have demonstrated that by combining specific surfactants in specific amounts and ratios, the adverse effects that surfactants have on beneficial microorganisms can be overcome, thereby enabling the viability, growth, and bioactivity of the microorganisms. The use of these surfactant blends in microbial-enhancing compositions maintains the viability of beneficial microorganisms while simultaneously advancing the United Nations Sustainable Development Goals ("SDGs"). Many bio-based surfactants are antagonistic to beneficial microorganisms, and the specific surfactant blends described herein enable the broader use of such bio-based surfactants in a variety of different applications. Bio-based surfactants are derived from renewable resources, and beneficial microorganisms such as Bacillus species are produced through fermentation, a less energy-intensive bio-based manufacturing process that uses renewable resources to produce biodegradable waste products with reduced environmental impact. These benefits also align with SDG Goal 12 (Responsible Consumption and Production). [Overview of the Initiative]

[0008] In one embodiment, the present technology provides a composition for overcoming antagonistic surfactant incompatibility between a surfactant and beneficial microorganisms, wherein antagonistic surfactant incompatibility adversely affects the viability, growth, or biological activity of beneficial living microorganisms, the composition comprising (a) at least one anionic surfactant in an amount of 0.1% to about 15% by weight of the active ingredient, which, alone, exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms, wherein the anionic surfactant is an alkyl sulfate, alkyl ether sulfate, alkyl sarcosinate, alpha-sulfonated alkyl ester, alkyl glutamate, and combinations thereof. (b) an anionic surfactant selected from the group consisting of (i) an anionic surfactant; (b) at least one further surfactant in an amount of 0.1% to about 15% by weight of the active ingredient, which, when used alone, exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms, wherein the further surfactant is selected from the group consisting of (i) amphoteric surfactants and (ii) glyceryl monoesters and / or glyceryl diesters; (c) optionally, at least one nonionic surfactant which, when used alone, exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms; (d) at least 10 4 (e) a carrier in an amount equal to 100% by weight of the composition in total, comprising an anionic surfactant, and further surfactants, and optionally a nonionic surfactant, when combined in the composition, overcomes antagonistic surfactant incompatibility between the surfactant and the beneficial microorganism and promotes the viability, growth, and biological activity of the beneficial microorganism.

[0009] In a further embodiment, the present technology provides a method for overcoming antagonistic surfactant incompatibility between a surfactant and beneficial microorganisms, wherein antagonistic surfactant incompatibility adversely affects the viability, growth, or biological activity of beneficial living microorganisms. The method comprises (a) at least 10 4(b) a step of providing beneficial microorganisms in cfu / mL; (c) a step of providing an anionic surfactant in an amount of 0.1% to about 15% by weight of the active ingredient, which alone exhibits antagonistic surfactant incompatibility when in contact with beneficial microorganisms and adversely affects the viability, growth, or biological activity of beneficial microorganisms, wherein the anionic surfactant is selected from the group consisting of alkyl sulfates, alkyl ether sulfates, alkyl sarcosinates, alpha-sulfonated alkyl esters, alkyl glutamates, and combinations thereof; (d) a step of providing a further surfactant in an amount of 0.1% to about 15% by weight of the active ingredient, which alone exhibits antagonistic surfactant incompatibility when in contact with beneficial microorganisms and adversely affects the viability, growth, or biological activity of beneficial microorganisms, further The surfactant comprises the steps of: (i) selecting from the group consisting of amphoteric surfactants and (ii) glyceryl monoesters and / or glyceryl diesters; (d) optionally providing a nonionic surfactant that, when alone, exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms; and (e) mixing beneficial microorganisms, anionic surfactants, further surfactants, and optionally nonionic surfactants together in a liquid carrier to form a composition, wherein the anionic surfactants, further surfactants, and optionally nonionic surfactants, when combined, overcome antagonistic surfactant incompatibility between the surfactants and beneficial microorganisms and promote the viability, growth, and biological activity of beneficial microorganisms.

[0010] In a related aspect, the technology provides a method for producing compositions that promote the viability, growth, and biological activity of beneficial microorganisms, the method comprising (a) at least 10 4(b) a step of providing beneficial live microorganisms in a concentration of cfu / mL; (c) a step of providing an anionic surfactant in an amount of 0.1% to about 15% by weight of the active ingredient, which, when used alone, exhibits antagonistic surfactant incompatibility when in contact with beneficial live microorganisms and adversely affects the viability, growth, or biological activity of the beneficial live microorganisms, wherein the anionic surfactant is selected from the group consisting of alkyl sulfates, alkyl ether sulfates, alkyl sarcosinates, alpha-sulfonated alkyl esters, alkyl glutamates, and combinations thereof; (d) a step of providing a further surfactant in an amount of 0.1% to about 15% by weight of the active ingredient, which, when used alone, exhibits antagonistic surfactant incompatibility when in contact with beneficial live microorganisms and adversely affects the viability, growth, or biological activity of the beneficial live microorganisms, further The surfactant comprises the steps of: (i) selecting from the group consisting of amphoteric surfactants and (ii) glyceryl monoesters and / or glyceryl diesters; (d) optionally providing a nonionic surfactant that, when alone, exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or biological activity of beneficial living microorganisms; and (e) combining beneficial microorganisms, anionic surfactants, further surfactants, optionally nonionic surfactants, and water under conditions sufficient to form a composition, wherein the anionic surfactants, further surfactants, and optionally nonionic surfactants, when combined, overcome antagonistic surfactant incompatibility between the surfactants and beneficial microorganisms and promote the viability, growth, and biological activity of the beneficial microorganisms.

[0011] In another aspect, the present technology provides a composition comprising an anionic surfactant at about 2.0 wt% to about 15 wt% based on the weight of the active ingredient; an amphoteric surfactant at about 2.0 wt% to about 15 wt% based on the weight of the active ingredient; optionally, an alcohol alkoxylate at 0 wt% to about 2.0 wt% based on the weight of the active ingredient, or alternatively, an alcohol alkoxylate at 0.1 wt% to about 2 wt% based on the weight of the active ingredient; optionally, a dialkylamide or an alkyl lactyl lactate at 0 wt% to about 2 wt% based on the weight of the active ingredient, or alternatively, a dialkylamide or an alkyl lactyl lactate at about 0.1 wt% to about 2 wt% based on the weight of the active ingredient; optionally, MgCl2 or NaCl at 0 wt% to about 7.0 wt% based on the weight of the active ingredient, or alternatively, MgCl2 or NaCl at 0.1 wt% to about 5 wt% based on the weight of the active ingredient; at least 10 4 cfu / mL of beneficial microorganisms; and water in an amount such that the total is 100 wt% of the composition. In some embodiments, the anionic surfactant comprises an alkyl ether sulfate, and the amphoteric surfactant comprises an alkylamidopropyl betaine.

[0012] In a further aspect, the present technology provides a composition comprising an alpha-sulfonated alkyl ester at about 2 wt% to about 7 wt% based on the weight of the active ingredient, an alkyl sarcosinate at about 0.2 wt% to about 2 wt% based on the weight of the active ingredient, a glyceryl monoester and / or a glyceryl diester at about 5 wt% to about 15 wt% based on the weight of the active ingredient, at least 10 4 cfu / mL of beneficial microorganisms; and water in an amount such that the total is 100 wt% of the composition.

[0013] In yet a further aspect, the present technology provides a composition comprising an alcohol alkoxylate having a hydrophilic-lipophilic balance (HLB) value of less than 10 at about 8 wt% to about 20 wt% based on the weight of the active ingredient, or alternatively, at about 8 wt% to about 15 wt% based on the weight of the active ingredient, an alkylamine oxide at about 2 wt% to about 5 wt% based on the weight of the active ingredient, an alkyl sarcosinate at about 0.2 wt% to about 2 wt% based on the weight of the active ingredient, at least 10 4 cfu / mL of beneficial microorganisms; and water in an amount such that the total is 100 wt% of the composition.

[0014] In some embodiments, the composition may be in the form of a liquid concentrate that is diluted, prior to use, at dilution rates of, inter alia, 1:1000, 1:400, 1:100, 1:64, 1:32, 1:16, or 1:10. In other embodiments, the composition may be a ready-to-use (RTU) composition in which the active ingredient is in an amount suitable for use.

Brief Description of the Drawings

[0015] [Figure 1] Figure 1 shows the compatibility profiles of different surfactants and surfactant blends against Bacillus spores identified using the resazurin assay. [Figure 2] Figure 2 is a graph showing the effect of spore addition on the viscosity of a surfactant blend. [Figure 3] Figure 3 is a contour plot showing the results of viscosity tests in a composition containing a blend of sodium lauryl ether sulfate (SLES) and cocamidopropyl betaine (CAPB). [Figure 4] Figure 4 is a contour plot showing the results of viscosity tests in a composition containing a blend of SLES and CAPB, as well as different amounts of dialkylamide and sodium chloride. [Figure 5] Figure 5 is a graph showing the dynamic viscosities of two different spore-containing compositions. [Figure 6] Figure 6 is a photograph showing the stability of a representative composition of the present technology and a comparative composition. [Figure 7] Figure 7 is a graph and photograph showing the sedimentation behavior of a representative composition of the present technology over a three-month period. [Figure 8] Figure 8 is a microscopic image showing the spore dispersibility of a composition of the present technology and a comparative composition. [Figure 9] Figure 9 is a graph showing the dynamic viscosity as a function of concentration in a formulation containing an additive of either dialkylamide or alkyl lactyl lactate. [Figure 10] Figure 10 is a contour plot showing the results of viscosity tests in a composition containing a blend of alkyl sarcosinate and alkyl betaine. [Figure 11] Figure 11 is a photograph comparing the biological activity of test compositions containing microbial spores, when combined with sodium lauryl sulfate alone, when combined with amine oxide alone, and when combined with a surfactant blend of sodium lauryl sulfate and amine oxide. [Figure 12] Figure 12 is a graph showing the viscosity of structured surfactant compositions with and without bacterial spores as a function of shear rate. [Figure 13] Figure 13 is a graph showing the yield stress of structured surfactant compositions with different weight ratios of low hydrophilic-lipophilic balance ("HLB") surfactants and high HLB surfactants, as well as different amounts of bacterial spores. [Modes for carrying out the invention]

[0016] definition "Beneficial microorganisms" refer to microorganisms that have a positive effect on the health and well-being of living organisms and ecosystems.

[0017] "Bioactivity" refers to the various processes and functions that microorganisms perform in their environment, including their ability to grow, replicate, metabolize nutrients, release enzymes and metabolites, move or bind molecules internally, generate and respond to signals, facilitate the cycling and availability of nutrients, and interact with other organisms.

[0018] "Antagonistic surfactant incompatibility" refers to surfactants that adversely affect the survival ability, growth, and biological activity of beneficial microorganisms. "Adverse effect" or "adverse effect" means interference with the biological capabilities (such as germination), growth capacity, or biological activity of beneficial microorganisms. Such interference includes interference with any process in the microbial life cycle, such as spore formation or germination of spores and conidia, production of metabolites and enzymes for digesting food sources, metabolism of these food sources, and maintenance and / or growth of beneficial microbial populations.

[0019] "Microbial-enhanced composition" means a composition comprising at least one surfactant that provides the main benefits of reducing aqueous surface tension and wetting substrates such as soil, leaves, or hard and soft surfaces, and at least one beneficial live microorganism.

[0020] "Inhibitory surfactants" refer to surfactants that, when used in a manner that reduces aqueous surface tension and results in wetting of substrates such as soil, leaves, and hard and soft surfaces, adversely affect the germination, growth, and / or biological activity of added beneficial microorganisms when used or combined in a common composition.

[0021] "Overcoming surfactant incompatibility" means the ability of an inhibitory surfactant blend to promote the biological activity of beneficial microorganisms in the presence of the inhibitory surfactant blend, as indicated by a decrease in droplet size or the appearance of turbidity after 2 to 6 days, as determined according to the test methods described in the Examples.

[0022] As used herein, "carbohydrates" include, but are not limited to, simple sugars and complex molecules, monosaccharides and disaccharides including glucose, fructose, galactose, sucrose, lactose, maltose, and xylose; polysaccharides such as starch, fiber, maltodextrin, amylose, amylopectin, and glycogen; and complex sugar sources such as molasses.

[0023] The Bio-Renewable Carbon Index (BCI) refers to the calculation of the percentage of carbon derived from bio-renewable resources, and is calculated based on dividing the number of bio-renewable carbons by the total number of carbons in the entire molecule.

[0024] "Bio-renewable" is defined herein as derived from animal, plant, or marine biological materials. The terms "active ingredient," "active ingredient %," and "active ingredient by weight %" refer to the amount of the active ingredient, without considering the amount of water or other solvents that may be present with the ingredient.

[0025] The terms "ready to use" or "RTU" products, compositions, or formulations of this technology mean products, compositions, or formulations that can be applied or used immediately as is. The “Dilution,” “Concentrate,” or “Dilution Concentrate” products, compositions, or formulations of this technology mean products, compositions, or formulations that need to be diluted with a diluent (e.g., water) in a ratio of, for example, 1:100, 1:64, 1:32, 1:16, or 1:10 before they can be applied or used for their intended purpose.

[0026] This technology is based on the discovery that many useful and important classes of surfactants are antagonistic to beneficial microorganisms at surfactant usage levels considerably lower than those required for typical product performance. However, it has been found that by combining specific surfactants that exhibit antagonistic surfactant incompatibility on their own, this surfactant incompatibility can be overcome, making those surfactants usable in compositions containing beneficial microorganisms. As a result, beneficial microorganisms in the composition can germinate, grow, and exhibit biological activity when specific blends of these incompatible surfactants are used in specific amounts and ratios. While not bound by any particular theory, it is believed that the desired effect of overcoming antagonistic surfactant incompatibility can be achieved by combining specific incompatible surfactants in selected amounts and ratios.

[0027] The incompatibility between surfactants and beneficial microorganisms has been investigated using various methods, including the standard zone of inhibition test, the standard agar plate test, or the standard resazurin (7-hydroxy-10-oxidephenoxazine-10-ium-3-one, sodium) (blue fluorescent dye) compatibility assay. In the zone of inhibition test, microorganisms are grown, cultured, diluted to the target level, and then applied to a petri dish containing agar nutrient medium. The test sample containing the target surfactant is diluted and packed into agar wells prepared in the agar nutrient medium. The plate is incubated to allow microbial growth to proceed. The test sample diffuses through the agar, and if the surfactant in the test sample has an inhibitory effect on the microorganism, a ring-shaped area of ​​non-growth is observed around the well filled with the sample. This ring-shaped area is called the zone of inhibition. The larger the zone of inhibition, the greater the inhibitory effect. The main limitations of this test method for non-antibiotic test samples are the inherent dilution of the active ingredient, the challenges of agar diffusion, and the unsuitability of the nutrient medium (neutralization, precipitation, etc.). While this method has been very well used in the medical field for antibiotics for decades, its suitability for testing non-antibiotic samples is low. In the standard agar plate test, a test sample containing the target surfactant and microorganism is exposed, diluted by several orders of magnitude, and incubated on nutrient agar. After an incubation period appropriate for the bacterial species, the microbial growth pattern is evaluated against a suitable control. This method is suitable for evaluating viable cell counts over time, but is not very suitable for inhibition testing due to the significant serial dilution required to remove all co-components from the test microbial species on the evaluation agar plate. In the resazurin-based test, a test sample containing the target surfactant is mixed with reduced microbial nutrients, beneficial microorganisms, and resazurin in a suspension. The test sample is incubated, and then examined for colorimetric and fluorescence changes. A blue test sample with low fluorescence indicates no biological activity and incompatibility between the surfactant and microorganisms. A pink test sample with high fluorescence indicates biological activity and compatibility between the surfactant and microorganisms.This test and other similar suspension tests address the limitations of the conventional tests mentioned above and provide a suitable, high-throughput, microorganism-independent platform for evaluating the inhibitory effects of surfactants on biologics.

[0028] Surfactants found to be incompatible with beneficial microorganisms at surfactant concentrations of 1 g / L or less include some anionic surfactants, some nonionic surfactants, most amphoteric surfactants (if not all), and most cationic surfactants (if not all). Specific incompatible surfactants include alkyl sulfates, alkyl ether sulfates, alkyl sarcosinates, alkyl glutamates, alpha-sulfonated alkyl esters, alkyl sulfonates, alkyl sulfoacetates, some alkyl phosphate esters, sulfosuccinates, rhamnolipids, hydroxysultaines, alkyl betaines, alkylamidopropyl betaines, alkylamine oxides, alkylamine alkoxylates, quaternized alkylamine alkoxylates, some alcohol alkoxylates, monoglycerides and / or diglycerides, and some EO / PO block copolymers. Surprisingly, however, by blending selected incompatible surfactants together in selected amounts and ratios, surfactant blends compatible with beneficial microorganisms can be produced, thereby allowing the incompatible surfactants to be used in compositions with beneficial microorganisms. A blend of incompatible surfactants comprises at least one incompatible anionic surfactant and at least one further surfactant that is incompatible with beneficial microorganisms. The further incompatible surfactant may be an amphoteric surfactant, or a glyceryl monoester and / or glyceryl diester, or a combination thereof. The blend may optionally also contain at least one nonionic surfactant that is incompatible with beneficial microorganisms.

[0029] Examples of anionic surfactants that can be used in the blend of surfactants include alkyl sulfates, alkyl ether sulfates, alpha-sulfonated alkyl esters, alkyl sarcosinates, and alkyl glutamates, including their sodium salts, potassium salts, magnesium salts, ammonium salts, monoethanolammonium salts, diethanolammonium salts, or triethanolammonium salts. Preferably, the anionic surfactant is derived from a natural source, and the BCI is at least 80, alternatively at least 90, alternatively at least 95, preferably 100. Examples of incompatible anionic surfactants for use in the compositions of the present technology are sodium lauryl sulfate, sodium lauryl ether sulfate, especially those having 1, 2, or 3 moles of ethylene oxide, sulfonated methyl C 12 ~C 18 ester (mono)sodium and sulfonated C 12 ~C 18 fatty acid (di)disodium, sodium lauroyl sarcosinate, and sodium cocoyl glutamate. Combinations of incompatible anionic surfactants can also be used. In one embodiment, the composition of the present technology can include, as the anionic surfactant component, a combination of an alpha-sulfonated alkyl ester and an alkyl sarcosinate. The composition of the present technology can include an anionic surfactant with an active ingredient amount of at least 0.1% by weight, such as from 0.1% to about 15% by weight. In some embodiments, the amount of the anionic surfactant can be within the range of from about 2% to about 15% by weight of the active ingredient amount, alternatively from about 2% to about 10% by weight of the active ingredient amount.

[0030] Examples of amphoteric surfactants that can be used in surfactant blends include alkylamine oxides, alkyl betaines, alkylamidopropyl betaines, and alkylsultaines. Preferably, the amphoteric surfactants are of natural origin, and the BCI is at least 80, alternatively at least 90, alternatively at least 95, and preferably 100. Examples of unsuitable amphoteric surfactants include lauramine oxide, cetyl betaine, cocoamidopropyl betaine, and cocoamidopropyl hydroxysultaine. Compositions of the present technology may contain amphoteric surfactants in an active ingredient amount of 0% to about 15% by weight. If a composition contains amphoteric surfactants, the amount of amphoteric surfactant is at least 0.1% by weight, such as 0.1% to about 15% by weight. In some embodiments, the amount of amphoteric surfactant may be in the range of an active ingredient amount of about 1% to about 15% by weight, alternatively about 1% to about 10% by weight, or alternatively about 2% to about 10% by weight.

[0031] Examples of incompatible nonionic surfactants that can be used in surfactant blends include alcohol alkoxylates, preferably alcohol ethoxylates, or glyceryl esters. Alcohol ethoxylates may be linear or branched and may have an HLB value of about 5 to about 15. Specific examples of unsuitable alcohol ethoxylates include BIOSOFT® EC 639, a linear alcohol ethoxylate with 8 moles of ethylene oxide; BIOSOFT® N23-3, a C12-C13 semilinear alcohol ethoxylate with 3 moles of ethylene oxide; BIOSOFT® N1-7, a C11 semilinear alcohol ethoxylate with 7 moles of ethylene oxide; MAKON® DA-6, a C10 branched alcohol ethoxylate with 6 moles of ethylene oxide; MAKON® UD-5, a C11 branched alcohol ethoxylate with 5 moles of ethylene oxide; and MAKON® DA-4, a C10 branched alcohol ethoxylate with 4 moles of ethylene oxide. All are available from Stepan Company, Northfield, Illinois. Glyceryl esters may be monoglycerides and / or diglycerides, or combinations thereof. A specific example of an unsuitable glyceride surfactant is STEPAN-MILD® GCC, which is a glyceryl caprylate / caprate mono- and di-glyceride surfactant. The amount of nonionic surfactant in the composition may be in the range of 0% to about 15% by weight of active ingredients, or alternatively, 0% to about 13% by weight of active ingredients.

[0032] Specific blends of incompatible anionic surfactants and incompatible amphoteric surfactants found to be compatible with beneficial microorganisms include blends of alkyl ether sulfates with alkylamidopropyl betaine and / or alkyl betaine, blends of alkyl sulfates with alkylamine oxides, and blends of alkyl sarcosinates with alkyl betaine. The weight ratio of anionic surfactant to amphoteric surfactant in the blend may be in the range of 5:1 to 1:2 based on the active ingredient level. Specific blends of incompatible anionic surfactants and incompatible nonionic surfactants include blends of alpha-sulfonated alkyl esters with glyceryl alkyl esters, where the weight ratio of nonionic surfactant to anionic surfactant is in the range of approximately 2:1 to approximately 2.5:1 based on the active ingredient level.

[0033] The total amount of surfactant in a composition varies depending on whether the composition is a concentrate intended to be diluted with water or other diluents before use, or whether the composition is a ready-to-use composition where the active ingredient is already at its final use concentration. In a concentrated composition, the total amount of surfactant may range from about 3% to about 20% by weight of the active ingredient, based on the total weight of the composition. In the case of a ready-to-use composition, the amount of surfactant may range from 0.1% to about 1% by weight of the active ingredient, based on the total weight of the composition.

[0034] In addition to overcoming antagonistic surfactant incompatibility and promoting the viability, growth, and biological activity of beneficial microorganisms, the surfactant blend in the composition of this technology unexpectedly improves the stability and dispersibility of beneficial microorganisms in the composition and prevents or minimizes spore aggregation. As used herein, stability means not only the ability of the composition to maintain spore viability without premature germination during storage, but also the ability of the composition to maintain beneficial microorganisms, particularly microbial spores, in a suspended state without precipitation. It has been found that incorporating beneficial microorganisms into a surfactant composition can significantly reduce the viscosity of the composition, resulting in a microbial-enhanced composition that cannot maintain the microorganisms in a stable suspended state. Consequently, the composition fails to achieve the desired enhancement of product performance expected from the addition of beneficial microorganisms.

[0035] Remarkably, the compositions of this technology provide an improved viscosity profile that allows beneficial microorganisms to be maintained in a suspended state without precipitation. The viscosity profile of a composition can be determined by measuring the viscosity as a function of shear rate at ambient temperature. A preferred viscosity profile is one in which the composition has high viscosity at low shear rates to suspend spores in the composition and low viscosity at high shear rates to facilitate handling of the composition. The compositions of this technology preferably have a viscosity of about 8,000 cP to about 30,000 cP when measured using a viscometer at 6 rpm and ambient temperature (about 25°C), and about 3,000 cP to about 8,000 cP when measured using a viscometer at 60 rpm and ambient temperature. The compositions of this technology have viscosities within both these low-shear and high-shear ranges and can maintain spores in a suspended state without significant separation, sedimentation, or deposition for at least one month, alternatively at least two months, preferably at least three months, and more preferably at least six months. The improved stability is at least in part due to the surfactant blend described herein' ability to increase viscosity and maintain the spores in a dispersed state without causing spore aggregation or aggregation.

[0036] The physical stability of a composition can be evaluated using various methods known in the art. For dilution concentrates, shelf life can be evaluated using Turbiscan (multiple light scattering) method, along with the average particle size of microbial spores as a function of storage time. For ready-to-use (RTU) compositions, the dispersibility of microbial spores in the composition can be evaluated using dynamic light scattering method, along with the average particle size and quantitative particle size distribution (polydispersity) of microbial spores. The formation of spore aggregation or aggregation can be monitored by optical microscopy for both dilution concentrates and ready-to-use (RTU) compositions, along with the qualitative particle size distribution of spores.

[0037] In some embodiments, surfactant blends can form structured surfactant systems that enable the maintenance of spores in a suspended state. As used herein, the terms “structured system” or “structured surfactant system” mean a fluid composition comprising water, a surfactant, and optionally other dissolving substances that together form an intermediate phase, or a dispersion of an intermediate phase in a continuous aqueous medium, which, when the system is at rest, has the ability to immobilize non-colloidal, water-insoluble particles, thereby forming a stable fluid suspension. The surfactant and water interact to form a phase that is neither liquid nor crystalline, which is usually referred to as a “liquid crystal phase,” or alternatively, an “intermediate state phase” or “intermediate phase.” Structured surfactant systems are generally known in the art and are described in more detail, for example, in U.S. Patent No. 9,668,474, which is incorporated herein by reference. Surfactants that can be used to prepare structured surfactant systems typically include a mixture of at least one surfactant having a low HLB value, e.g., less than 10, or alternatively less than 9, and at least one surfactant having a high HLB value, e.g., 10 or more. Specific surfactant blends that enable both the formation of a structured surfactant system for suspending microbial spores in a stable suspension and the overcoming of antagonistic surfactant incompatibility include alpha-sulfonated alkyl esters combined with glyceryl caprylate / caprate esters and alkyl sarcosinates, as well as alkylamine oxides combined with low-HLB ethoxylated fatty alcohols and alkyl sarcosinates.

[0038] In some embodiments, it may be desirable to include inorganic salts and / or certain dialkylamides or certain alkyllactyl lactylates in the compositions of the Technology. It has been found that by adding certain inorganic salts and / or suitable dialkylamides or alkyllactyl lactylates in specific amounts, the viscosity of the composition can be increased and the stability and dispersibility of beneficial microorganisms in the composition can be improved. Suitable inorganic salts include sodium chloride and magnesium chloride (or their hydrates). An example of a suitable dialkylamide is NINOL® CAA ("CAA"), a mixture of dimethyl lauramide and dimethyl myristamide available from Stepan Company. CAA is derived primarily from renewable sources and has a BCI of 86. An example of a suitable alkyllactyl lactate is STEPAN-MILD® L3 ("L3"), a lauryl lactylate with a BCI of 100, available from Stepan Company. When added, the amount of inorganic salt, dialkylamide, or alkyl lactyl lactate in the composition depends on the specific surfactant and the amount of the specific surfactant in the composition. The amount of sodium chloride or magnesium chloride in the composition may be in the range of 0% to 7% by weight, or alternatively, about 1% to about 5% by weight relative to the composition. The amount of dialkylamide or alkyl lactyl lactate in the composition may be in the range of 0% to about 2% by weight as an active ingredient, or alternatively, about 0.1% to about 2% by weight as an active ingredient, based on the total weight of the composition. The weight ratio of anionic and amphoteric surfactants to dialkylamide or alkyl lactyl lactate may be in the range of about 10:1 to about 25:1, preferably in the range of about 15:1 to about 25:1.In one embodiment, a composition having improved stability and dispersibility, as well as a good viscosity profile, comprises about 2.0% to about 15% by weight of an alkyl ether sulfate anionic surfactant, about 2.0% to about 15% by weight of an alkylamidopropyl betaine amphoteric surfactant, 0% to about 2% by weight of an alkylamidopropyl betaine amphoteric surfactant, alternatively, about 0.1% to about 2% by weight of an alcohol alkoxylate nonionic surfactant, alternatively, about 0% to about 2% by weight of an alcohol alkoxylate nonionic surfactant, alternatively, about 0.1% to about 2% by weight of an alcohol alkoxylate or alkyl lactyl lactate, 0% to about 7.0% by weight of an alcohol alkoxylate, alternatively, about 1% to about 5% by weight of an alcohol alkoxylate or sodium clitrate, at least 1 × 10⁻¹⁶. 4 It contains beneficial microorganisms in CFU / g amounts, and water in an amount totaling 100% by weight of the composition.

[0039] The beneficial microorganisms that can be used in the compositions of this technology may be bacteria, fungi, yeasts, or molds, and may be endospores (also called spores), vegetative cells, conidial cells, or mycelial cells. In some embodiments, the bacterial endospores are of the genus Bacillus. Bacillus spores include non-pathogenic mutants of Bacillus cereus such as Bacillus amyloliquefaciens, Bacillus brevis, and toyoi, as well as Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus halodurans, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, and Bacillus methylotrophicus. It may be one or more of the following: Bacillus methylotrophicus, Bacillus mycoides, Bacillus pasteurii, Bacillus polyfermenticus, Bacillus polymyxa, Bacillus pumilus, Bacillus simplex, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thiaminolyticus, Bacillus thuringiensis, and combinations thereof.In some embodiments, the beneficial microorganisms are a blend of bacterial spores from two or more strains, particularly a blend of two or more Bacillus strains. Blends of Bacillus spores from different Bacillus species are commercially available from various sources. A blend of Bacillus spores may include spores from one or more of the following: Bacillus subtilis, Bacillus amyloricephaciens, Bacillus licheniformis, Bacillus megatherium, Bacillus thuringiensis, and Bacillus pumilus. The amount of beneficial microorganisms in a composition of this technology varies depending on the end use of the composition, but is generally at least 1 × 10⁻⁶. 4 CFU / g, more preferably at least 1 × 10⁻¹⁶ 5 The value is CFU / g. If the composition is a concentrate that is diluted before use, the amount of beneficial microorganisms is 1 × 10⁻⁶. 7 ~1 × 10 10 CFU / g, preferably 1 × 10⁻⁶ 9 ~5×10 9 It may be within the range of CFU / g. If the composition is a ready-to-use composition, the amount of beneficial microorganisms is 1 × 10⁻⁶ 4 ~1 × 10 9 CFU / g, another option is 1 × 10 5 ~1 × 10 9 It may be within the range of CFU / g.

[0040] The composition of this technology may also include additives that can enhance the ability of surfactant blends to promote the viability, growth, and metabolic activity of beneficial microorganisms. The additives may include one or more of L-amino acids, inorganic divalent metal salts, or monovalent salts. The additives may also include carbohydrates as an optional component.

[0041] The L-amino acid may be one or more L-amino acids selected from the group consisting of L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-valine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-serine, L-arginine, L-cysteine, L-glutamine, L-glycine, L-proline, and L-tyrosine. In some embodiments, the L-amino acid is L-alanine.

[0042] The inorganic divalent metal salt may be one or more of the following: magnesium chloride, calcium chloride, manganese chloride, iron chloride, copper chloride, zinc chloride, cobalt chloride, magnesium nitrate, magnesium sulfate, calcium sulfate, manganese sulfate, iron sulfate, copper sulfate, zinc sulfate, cobalt sulfate, magnesium citrate, calcium citrate, any of the above hydrates, or any combination thereof. In some embodiments, the divalent metal salt is magnesium chloride or its hydrate.

[0043] The monovalent salt may be one or more of the following: potassium nitrate, sodium nitrate, potassium chloride, potassium iodide, potassium manganese oxide, potassium sulfate, sodium bicarbonate, sodium sulfate, ammonium nitrate, ammonium sulfate, ammonium chloride, sodium citrate, potassium citrate, ammonium citrate, or a combination thereof. In some embodiments, the monovalent metal salt is potassium nitrate.

[0044] The additive may optionally contain carbohydrates, which can provide a food source for beneficial microorganisms and enhance their growth. The carbohydrates may be one or more of glucose, maltose, galactose, fructose, sucrose, lactose, molasses, glycogen, or glucan. In some embodiments, the carbohydrate is glucose. If carbohydrates are included in the additive, their amount is preferably less than or equal to the total weight of the other additive components in the composition (L-amino acids, divalent metal salts, and / or monovalent salts).

[0045] The compositions of this technology may contain additional optional components depending on the end use of the composition. Such other components include additional surfactants compatible with beneficial microorganisms, hydrotropes or other solubilizers for obtaining and maintaining a clear, single-phase concentrated composition or a ready-to-use composition, carbohydrates as a supplemental food source for beneficial microorganisms, builders, pH adjusters, electrolytes to enhance the cleaning power of surfactants, enzymes to improve cleaning properties, fragrances for various attractive scents, dyes for preferred colors, preservatives, and other functional components. Preferably, any of the optional components are compatible with beneficial microorganisms.

[0046] Surfactants that have been found to be compatible with beneficial microorganisms and may be included in the composition include, but are not limited to, castor oil ethoxylate, alpha-olefin sulfonates, such as BIO-TERGE® AS-40 of C14-16 olefin sulfonates, polyoxyethylene sorbitan monooleates, such as those sold under the trademarks TWEEN® and SPAN®, certain alkyldimethylamides, such as STEPOSOL® MET-10U of unsaturated C10 N,N-dimethylamide, and combinations thereof.

[0047] Suitable hydrotropes for use in the compositions of this technology include sodium xylenesulfonate, cumenesulfonate, amphoteric dipropionate salts, and combinations thereof. Suitable carbohydrates for use as supplemental food sources include cellulose, maltodextrin, fiber, amylose, amylopectin, glycogen, starch, or combinations thereof. Sodium gluconate and sodium citrate dihydrate are builders that have been found to be compatible with beneficial microorganisms.

[0048] To enhance the cleaning properties, enzymes may be included in the composition of this technology. Suitable enzymes include proteases, amylases, and lipases. Preservatives may be included in the composition of this technology, provided that the amount of preservative does not affect the viability, growth, and / or biological activity of beneficial microorganisms at the dilution ratios used in the composition. Determining the appropriate amount of preservative to be used in the composition requires a careful balance between achieving good efficacy against pathogenic bacteria and fungi and maintaining the viability of beneficial microorganisms in concentrated form and enabling their growth in a ready-to-use state. Not all preservatives can achieve this balance.

[0049] For example, 2-phenoxyethanol is a common preservative used in hard surface cleaning agent compositions. A typical concentration of 2-phenoxyethanol is about 0.2% by weight of the composition, and 1.5% by weight is usually sufficient to preserve most compositions. However, in some embodiments of this technology, 2-phenoxyethanol at a concentration of 1.5% by weight may not provide sufficient efficacy against pathogenic bacteria and fungi. Combining benzoic acid and 2-phenoxyethanol at a concentration of 1.5% by weight (0.75% 2-phenoxyethanol and 0.75% benzoic acid) provides good efficacy against pathogenic bacteria and fungi without affecting the viability, growth, and / or biological activity of beneficial microorganisms. Other preservatives that have been found to provide a balance between good efficacy against pathogenic bacteria and fungi and maintenance of the viability and performance of beneficial microorganisms include combinations of chloromethylisothiazolinone and methylisothiazolinone (e.g., Kathon CG), benzisothiazolinone, and benzoic acid. Other preservatives that may be considered for use include benzyl alcohol and fatty alcohols. Any combination of these preservatives may also be considered. The amount of preservative will vary, at least in part, depending on the specific preservative or combination of preservatives selected, but generally, it is desirable to use the smallest amount of preservative that provides the necessary efficacy against pathogenic bacteria and fungi.

[0050] The compositions of this technology are typically in liquid form and contain at least one carrier in an amount equal to 100% of the total composition. Water is a suitable carrier, particularly in ready-to-use formulations, and may be deionized water, hard water, soft water, distilled water, tap water, or a combination thereof. Water may be used alone as a carrier, or in combination with other suitable carriers, such as water-miscible solvents like alcohols or glycol ethers, provided that such solvents are compatible with beneficial microorganisms.

[0051] The compositions of this technology can be used in a variety of end uses. In particular, the compositions of this technology are intended to be used in any end use in which beneficial microorganisms and at least one incompatible anionic surfactant and at least one incompatible amphoteric surfactant are used, and the combination of surfactants overcomes antagonistic surfactant incompatibility. Such end uses include microorganism-enhanced detergents, including detergents for hard and soft surfaces, agricultural formulations, personal care formulations, bioremediation, wastewater treatment, fermentation, probiotics, animal health, aquaculture, water recycling, and food applications.

[0052] As an example, the compositions of this technology can be used as microbially enhanced cleaning compositions. When the compositions are used to clean surfaces, spores germinate and digest dirt that is often inaccessible during initial cleaning, such as grout, floors, and dirt embedded in porous surfaces such as countertops. The surfactant blend in the cleaning composition provides initial cleaning of the surface, while the combination of surfactants overcomes the antagonistic surfactant incompatibility that each surfactant would exhibit individually, thereby enabling the survival and proliferation of microbial spores and enhancing cleaning performance. It is assumed that microbially enhanced cleaning compositions using the surfactant blends described herein can be formulated without microbial spores, and microbial spores can be added to the composition at the time of use. Alternatively, the microbially enhanced compositions can also be formulated as complete microbially enhanced cleaning compositions containing a combination of microbial spores and surfactants together with any of the components.

[0053] Microbial-enhanced cleaning compositions may be formulated, for example, as ready-to-use products or concentrated products for dilution. Concentrated products may be up to 100 times the ready-to-use component levels. Whether in ready-to-use form or concentrated for dilution, the final usable concentration of the components is equivalent. The final usable concentration is approximately 0.05% to approximately 1% by weight of the surfactant active ingredient, or alternatively, approximately 0.1% to approximately 1% by weight of the surfactant active ingredient, and approximately 1 × 10⁻⁶ 4 ~Approx. 1×10 8 CFU / g, another option is approximately 1 × 10 5 ~Approx. 1×10 8 It may contain beneficial microorganisms in CFU / g. Dilution concentrates may contain approximately 3.0% to 20% by weight of surfactant active ingredient, or as an alternative, approximately 5% to 15% by weight of surfactant active ingredient, and approximately 1 × 10⁻⁶ 7 ~Approx. 1×10 10 It may contain beneficial microorganisms in CFU / g. In some embodiments, dilution compositions are preferred as cost-saving and expense-reducing options, as they reduce packaging and transport costs. In some embodiments, concentrates may be packaged as liquids or sprays that can be diluted to working concentrations on-site and used immediately.

[0054] The diluent for diluting the concentrated form of the composition may be any diluent system known in the art. Suitable diluents include, but are not limited to, water, glycols (preferably propylene glycol), alcohols (e.g., isopropanol, ethanol, methanol), other polar solvents known in the art, and mixtures thereof. Water is a preferred diluent for the techniques described in this invention and may be deionized water, hard water, soft water, distilled water, tap water, or a combination thereof.

[0055] As another example of end-use, the compositions of this technology can be used in agricultural bio-insecticides and bio-fungicides that utilize inhibitory anionic and amphoteric surfactants in combination with beneficial microorganisms. In agriculture, microorganisms, typically of the spore type and typically of the Bacillus genus, are applied to plants and / or fields to suppress the growth of pathogenic organisms, including fungi, and help crops grow more healthily. Many agricultural applications rely on surfactants to effectively wet the leaf surface for the efficient use of components in formulations containing microorganisms such as Bacillus. However, these agricultural surfactants can adversely affect the viability, growth, and / or bioactivity of microorganisms. Certain blends of anionic and amphoteric surfactants in the compositions of this technology can overcome the adverse effects of each surfactant, thereby enabling the use of these inhibitory surfactants in agricultural bio-insecticides and bio-fungicides.

[0056] For agricultural applications, the compositions of this technology are typically formulated as concentrates and diluted with water or other suitable diluents before use at dilution ratios ranging from 1:2.5 to 1:3200, alternatively 1:50 to 1:1500, alternatively 1:100 to 1:1000, or alternatively 1:256 to 1:512, preferably at a dilution ratio of 1:400. The concentrate contains about 1.0% to about 20% by weight of surfactant active ingredients and about 1 × 10⁻⁶ 7 ~Approx. 1×10 10 It may contain beneficial microorganisms in CFU / g.

[0057] This technology also includes a method for overcoming surfactant incompatibility between beneficial live microorganisms and surfactants, which adversely affects the viability, growth, or biological activity of beneficial live microorganisms. The method involves mixing beneficial live microorganisms in a liquid carrier such as water with a blend of at least one anionic surfactant exhibiting antagonistic surfactant incompatibility and at least one amphoteric surfactant or glyceryl monoester and / or glyceryl diester surfactant exhibiting antagonistic surfactant incompatibility, wherein the combination of anionic surfactant and amphoteric or glyceryl ester surfactant overcomes antagonistic surfactant incompatibility and promotes the viability, growth, and biological activity of beneficial microorganisms. The amount of anionic surfactant is in the range of 0.1% to 15% by weight of the active ingredient, and the amount of amphoteric glyceryl monoester and / or glyceryl diester is in the range of 0.1% to 15% by weight of the active ingredient. The weight ratio of anionic surfactant to amphoteric or glyceryl ester surfactant in the blend may be in the range of 5:1 to 1:2 based on the active ingredient level. In some embodiments, the method may further include adding a nonionic surfactant exhibiting antagonistic surfactant incompatibility to the anionic and amphoteric surfactants. In one embodiment of the method, beneficial microorganisms are mixed with the incompatible anionic surfactant (and nonionic surfactant, if present) in water in a preferred liquid carrier, and then the incompatible amphoteric surfactant is added and mixed to increase the viscosity of the composition. In another embodiment, if the amount of surfactant is the dilution ratio used in the liquid carrier, and the viscosity of the final composition may be as low as water, the beneficial microorganisms may be added after the surfactants have been mixed together to minimize precipitation of the beneficial microorganisms from the solution state. In further embodiments, the incompatible anionic surfactant and the incompatible glyceryl monoester and / or glyceryl diester are mixed in water in a first step to form a structured surfactant system, and the beneficial microorganisms in water are added to the structured surfactant system in a second step.

[0058] The pH of the composition may be adjusted to a range of 5.5 to 8, if necessary, depending on the selection of the unsuitable surfactant. Standard mixing equipment may be used to mix the unsuitable surfactant and beneficial microorganisms.

[0059] Those skilled in the art will recognize that modifications can be made to the present invention without departing from the spirit or scope of the invention. The present invention is further described by the following examples, but these examples should not be construed as limiting the spirit or scope of the invention to the specific procedures or compositions described therein.

[0060] The pH of the composition was determined at room temperature (20-25°C) using a calibration electrode. Using Stat-Ease 360 ​​software, sample sets for each experimental design were created, the test results were modeled from the obtained data, and statistical results for the optimal model were generated. Subsequently, optimization plots were created using this model and the numerical optimization function of Stat-Ease 360. The optimization included contour plots of the "desirability" of component levels against a defined design space. These plots visually represent the complex relationships between surfactant components and any additional components, showing areas where the desired performance is high, low, or zero.

[0061] In several examples, a resazurin-based assay was used to determine the compatibility of bacterial spores with surfactants or surfactant blends. This assay was performed by combining the surfactant or blend with bacterial spores, limited microbial base nutrients, and resazurin in water, and then incubating the test samples. The test samples were then examined for changes in colorimetry and fluorescence. Test samples that were blue and had low fluorescence indicated no biological activity and incompatibility between the surfactant and the microorganism. Test samples that were pink and had high fluorescence indicated biological activity and compatibility between the surfactant and the microorganism. [Examples]

[0062] Example 1: Suitability test of surfactants and surfactant blends The compatibility between beneficial microorganisms and different surfactants and surfactant blends was determined using a resazurin-based assay. Aqueous test compositions were prepared, containing different concentrations of the target surfactant in water, along with a restriction nutrient basal medium, a blend of Bacillus bacterial spores, and a resazurin dye. A surfactant was considered inhibitory if a test composition containing a surfactant concentration of less than 1.0 g / L maintained a blue color and did not fluoresce when compared to a suitable control. The surfactants tested included alkyl ether sulfate (STEOL® CS-270 Plus, 2EO of sodium lauryl ether sulfate (2 moles of ethylene oxide)) ("SLES"), alkylamidopropyl betaine (AMPHOSOL® CG-50, cocoamidopropyl betaine) ("CAPB"), alkyl sarcosinate (MAPROSYL 30-B, sodium lauroyl sarcosinate), alkyl glutamate (sodium cocoyl glutamate), and alcohol ethoxylate (BIO-SOFT® EC-639, 8 moles of ethylene oxide in lauryl alcohol) ("Laureth 8EO"). Two surfactant blends were also tested: a blend of SLES, CAPB, and Laureth 8EO, and a blend of SLES, CAPB, Laureth 8EO, and alkyldimethylamide (NINOL® CAA) ("CAA"). The results of the resazurin assay are shown in Figure 1. Short bar graphs indicate that the surfactant is inhibitory and unsuitable for Bacillus spores, while long bar graphs indicate that the surfactant is suitable for the spores. As shown in Figure 1, each surfactant individually was found to be inhibitory. Surprisingly, the blend of SLES, CAPB, and Laureth 8EO showed suitability for Bacillus spores at high surfactant concentrations (greater than 10 g / L), even though each of SLES, CAPB, and Laureth 8EO was inhibitory individually. Similarly, the blend of SLES, CAPB, Laureth 8EO, and CAA showed suitability for the spores at concentrations greater than 10 g / L, even though each of SLES, CAPB, and Laureth 8EO was inhibitory individually.These results indicate that a specific blend of inhibitory surfactants can overcome the inhibitory effect on Bacillus spores that would otherwise be present in each individual surfactant.

[0063] Example 2: Effect of spore addition on the viscosity of the composition Aqueous test compositions containing a blend of SLES anionic surfactant and CAPB amphoteric surfactant were prepared using different amounts of the surfactant blend. Each composition also contained 4.5% by weight of sodium chloride. Each composition was 2 × 10 9 Similar compositions were also prepared, except that they included a commercially available Bacillus spore blend at CFU / g. The dynamic viscosity of the compositions was measured at 6 rpm and ambient temperature. The viscosity data was plotted as a function of surfactant concentration, and the results are shown graphically in Figure 2. The graph in Figure 2 shows that, without spores, the composition has a viscosity of approximately 25,000 cP to approximately 46,000 cP, depending on the surfactant concentration. However, when spores were added, at the same surfactant concentration, the composition had a viscosity of only about 2,000 cP to approximately 6,000 cP. This result indicates that the viscosity of surfactant compositions can be significantly reduced when beneficial microorganisms are added.

[0064] Example 3: Composition comprising surfactant alkyl ether sulfate and alkylamidopropyl betaine Compositions were prepared using blends of the anionic surfactant alkyl ether sulfate (sodium lauryl ether sulfate 2EO) ("SLES") and the amphoteric surfactant alkylamidopropyl betaine (cocamidopropyl betaine) ("CAPB"), with different amounts of SLES and CAPB. All compositions contained 4.5% by weight of NaCl and 1 × 10⁻⁶ 6The samples contained a commercially available Bacillus spore blend at CFU / g. The dynamic viscosity of each composition was measured using a viscometer at 6 rpm and ambient temperature. Using the viscosity data, Stat-Ease 360 ​​optimized contour plots were created for the test compositions. The contour plots are shown in Figure 3. The contour plots show contours corresponding to different amounts of SLES and CAPB, indicating a desirable high viscosity region (20,000 cP or higher) (central region of the plot) and a less desirable low viscosity region (lower right and upper left regions of the plot). From the contour plots, it can be determined that the central region of the plot indicates a composition where high physical stability (stable suspension of spores) is expected. As shown in Figure 3, the high viscosity region occurs with specific amounts and weight ratios of SLES and CAPB. Useful amounts of SLES to obtain high viscosity may range from approximately 2.75% to approximately 11.5% by weight of active ingredient, and useful amounts of CAPB may range from approximately 2% to approximately 6% by weight of active ingredient. The weight ratio of SLES to CAPB can be in the range of approximately 20:1 to 1:1, as an alternative, 10:1 to 1:1, as an alternative, 5:1 to 1:1, as an alternative, or 3.0:1 to 1:1, and preferably in the range of approximately 2.7:1 to approximately 1:1.

[0065] Example 4: Composition comprising surfactant alkyl ether sulfate and alkylamidopropyl betaine, and optional components. Test compositions containing the surfactants SLES and CAPB, as well as different amounts of NaCl and the alkyldimethylamide NINOL® CAA ("CAA"), were prepared to evaluate whether the addition of NaCl and / or CAA could further increase the viscosity of the SLES / CAPB surfactant blend, particularly at lower surfactant concentrations. The viscosity of the test compositions was determined and used to create Stat-Ease 360 ​​optimized contour plots for the test compositions. The contour plots are shown in Figure 4. The optimized contour plots show contours corresponding to different amounts of NaCl and CAA in the compositions, representing a high-desirability region (center to right of the plot) and a low-desirability to zero-desirability region (left side, lower right corner, and upper right edge of the plot). The high-desirability region indicates that the maximum viscosity is produced with the combination of CAA and NaCl, but the amount of CAA should not exceed 2.5% by weight. The clear range from high desirability to zero desirability indicates that the viscosity of the composition can be increased by adding NaCl and CAA to the surfactant blend of SLES and CAPB, and further indicates that specific amounts of these components are important to achieve the viscosity necessary to maintain the physical stability of beneficial microorganisms in the composition.

[0066] Example 5: Viscosity profile of a composition containing alkyl ether sulfate, alkylamidopropyl betaine, and an optional component A microbially enhanced composition was prepared using SLES as the anionic surfactant, CAPB as the amphoteric surfactant, and a commercially available Bacillus spore blend as the beneficial microorganism. This formulation also contained CAA, NaCl, and BIOSOFT EC 639, an alcohol ethoxylate with 8 moles of ethoxylate per molecule, which is a nonionic surfactant incompatible with the beneficial microorganism. Two compositions were prepared, and their formulations are shown in Table 1.

[0067] [Table 1]

[0068] The viscosity of the two compositions was measured using a rheometer as a function of shear rate at 25°C. Figure 5 shows the viscosity profiles of the two compositions as a function of shear rate. Both compositions exhibit similar viscosities around 1000 cP at high shear rates, but composition A shows a high viscosity of around 12000 cP at low shear rates, meaning that this composition can suspend spores when at rest, but is also fluid at high shear rates. However, composition B shows a low viscosity of around 1000 cP even at low shear rates, indicating that this composition is not suitable for suspending spores.

[0069] Figure 6 shows the appearance of compositions A and B after one month of storage at ambient temperature. Composition A exhibits good physical stability with no precipitation, while composition B shows significant precipitation at the bottom of the glass container. The results in Figures 5 and 6 indicate that specific amounts and ratios of components are important to achieve the desired viscosity and stability properties. Here, the amounts of surfactants SLES and CAPB in composition B are insufficient to completely solubilize CAA, resulting in separation and low viscosity. The component amounts in composition B are close to the low-desirability region in the contour plots created in Examples 3 and 4. Increasing the amount of CAPB in composition B or adjusting the amount of CAA and / or NaCl are methods for adjusting the amounts and ratios of components to achieve the desired viscosity and stability properties.

[0070] Composition A has the following physical properties: Specific gravity: 1.03~1.05 g / cm³ 3 The analysis included the active ingredient content based on refractive index, Brix value: 13.0-18.0, pH (as is): 6.2-7.7, and viscosity measured using a viscometer at 25°C and 60 rpm: 4000-8000 cP.

[0071] Example 6: Spore dispersibility in a composition containing alkyl ether sulfate, alkylamidopropyl betaine, and a nonionic surfactant. The dispersion properties of composition A in Example 5 were evaluated. Using Turbiscan, the ability of composition A to maintain spore dispersibility over time without sedimentation was determined. After 3 months of storage at ambient temperature, composition A exhibited good physical stability without precipitation. Figure 5 shows the dispersibility results from Turbiscan graphically, and the photograph shows composition A's ability to maintain physical stability without precipitation. The Turbiscan results demonstrate that composition A of this technology is physically stable and can suspend bacterial spores over time without sedimentation.

[0072] Composition A was a concentrate, which was diluted 1:100 with DI water to obtain the RTU composition. The number of spores in the RTU composition was approximately 1.9 × 10⁶. 7 The concentration was CFU / g. Subsequently, the spore dispersibility in the RTU composition was characterized by dynamic light scattering. For comparison, a surfactant-free spore suspension (1.9 × 10⁻⁶) was used. 7 The CFU / g was prepared in DI water. Table 2 shows the dispersibility of these two compositions, characterized by average particle size, polydispersity, and particle number.

[0073] [Table 2]

[0074] Table 2 shows that the average spore size in the RTU formulation was smaller than that of the control, and the polydispersity was four times lower than that of the control. The number of particles in the RTU formulation was also higher than that of the control. The high average spore size and polydispersity, and the low number of particles in the control suspension, indicate the formation of spore aggregates. The results in Table 2 demonstrate that the RTU formulation of composition A of this technology provides good dispersibility of bacterial spores at RTU dilution levels without causing spore aggregation.

[0075] Microscopic image analysis of the RTU formulation was performed to observe the appearance of spores in the RTU formulation. A magnification of 40x objective lens was used to observe particles larger than 1 micrometer. Figure 8 shows the appearance of spores under an optical microscope. This image is compared to a control suspension containing spores without the surfactant blend of composition A. The RTU formulation does not show spore aggregates, while the spore suspension without the surfactant blend shows large spore aggregates. This observation is consistent with the results of dynamic light scattering shown in Figure 7. Overall, these results demonstrate that composition A of this technology provides good dispersibility of bacterial spores at both the concentrate level and the RTU level.

[0076] Example 7: Composition in which alkyldimethylamide is replaced with alkyllactyl lactate In this example, we evaluated whether similar viscosity and stability properties could be obtained by using alkyl lactyl lactate (STEPANMILD® L3) instead of dialkylamide (dimethyl lauramide / myristamide) in a formulation similar to composition A of Example 5. Test compositions were prepared using the formulations shown in Table 3, with one set of repeatable test compositions containing dialkylamide and the second set of repeatable test compositions containing alkyl lactyl lactate.

[0077] [Table 3]

[0078] The test compositions were prepared by adding 1.0% by weight of the formulations listed in Table 3 to water, with one set of test compositions using dialkylamide and the other set using alkyllactyl lactate. Each test composition also contained 1.0% by weight of 33 g / L nutritional mix (30 g / L tripty soy broth and 3 g / L yeast extract).

[0079] The test compositions were visually evaluated for bacterial spore germination and growth using the Sudan III-stained extra virgin olive oil (EVOO) test. EVOO is a food source for vegetative cells and, when added to test samples containing bacterial spores, can serve as an indicator of biological activity. Under favorable conditions, bacterial spores germinate and grow into fully functional vegetative cells that secrete enzymes capable of digesting EVOO. Microbial growth and biological activity are evident from the thinner, stronger red layer observed on top of the test sample due to the digestion of EVOO and the increasing concentration of undigested Sudan III dye. Growth and biological activity are also evident from the turbidity that develops in the test composition, which is also visually detectable. Test compositions that do not develop turbidity indicate little to no biological activity for microorganisms. In the stained EVOO test, 0.2% by weight of Sudan III (62.5 ppm)-stained EVOO was added to each test composition, and the pH was adjusted to 7 using L-lactic acid or dilute NaOH as needed. The test compositions were first photographed, and then photographed again after being shaken for four days in an orbital mixer set to "5" on a scale of 0 to 10. Formulations containing dialkylamide and alkyl lactyl lactate demonstrated good biological growth, indicating that the surfactant blend in the formulation was able to overcome the incompatibility between each individual surfactant and bacterial spores.

[0080] The dynamic viscosity of the dialkylamide and alkyllactyl lactate test compositions was measured at 6 rpm and ambient temperature. The viscosity data was plotted as a function of the concentrations of dialkylamide and alkyllactyl lactate in each composition, as shown in Figure 9. The graph in Figure 9 shows that the viscosities of the two formulations are equivalent, demonstrating that alkyllactyl lactate is substituted in a 1:1 ratio for dialkylamide to increase the viscosity of the composition and improve the stability and dispersibility of beneficial microorganisms in the composition.

[0081] Example 8: Composition comprising surfactants alkyl sarcosinate and alkyl betaine, and optional components. Aqueous compositions were prepared containing blends of alkyl sarcosinate (sodium lauroyl sarcosinate) anionic surfactant and alkyl betaine (cetyl betaine) amphoteric surfactant with varying amounts and ratios. All compositions were 1 × 10⁻⁶ 6 The composition contained a commercially available Bacillus spore blend at CFU / g. The dynamic viscosity of the composition was measured using a viscometer at 6 rpm and ambient temperature. Using the viscosity data, a Stat-Ease 360 ​​optimized contour plot was created for the test composition. The contour plot is shown in Figure 10. The contour plot shows contours indicating a high viscosity region (20,000 cP or higher) (left corner of the plot) and a low viscosity region (right corner of the plot), corresponding to different amounts of alkyl sarcosinate and cetyl betaine. From the contour plot, it can be determined that the region in the left corner of the plot indicates a composition in which high physical stability (stable suspension of spores) is expected. As shown in Figure 10, the high viscosity region occurs at specific amounts and weight ratios of the surfactant alkyl sarcosinate and alkyl betaine. The preferred weight ratio of alkyl sarcosinate to alkyl betaine is approximately 1:1 to approximately 1:2.

[0082] To evaluate the ability of surfactant blends of alkyl sarcosinate and alkyl betaine to overcome antagonistic incompatibility between each surfactant individually and bacterial spores, repeated test compositions were prepared. The test formulation consisted of 20% by weight (30% active ingredient) of sodium lauroyl sarcosinate and 32% by weight (30% active ingredient) of cetyl betaine, 100 × 10⁻⁶. 9The test composition contained 3.5% by weight of a commercially available Bacillus spore blend containing CFU / g of Bacillus spores, 4% by weight of potassium nitrate, lactic acid (88% active ingredient) to adjust the pH to 5.5-6.0, and the remainder being water. The test composition was prepared by diluting the test formulation with water to a strength of 0.3% and adding 2% by weight of a 3.3 g / L nutritional mix (3 g / L of tripty soy broth and 0.3 g / L of yeast extract). Bacterial spore germination and growth were visually evaluated using an unstained EVOO test. As described above, EVOO is a food source for vegetative cells and, when added to a test sample containing bacterial spores, can serve as an indicator of bioactivity. Under favorable conditions, bacterial spores germinate and grow into fully functional vegetative cells that secrete enzymes capable of digesting EVOO. Microbial growth and bioactivity are evident from the turbidity that develops in the test composition, which is detectable by visual inspection. Test compositions that do not produce turbidity indicate no or minimal biological activity against microorganisms. In the unstained EVOO test, 0.1% by weight of unstained EVOO was added to each test composition. Subsequently, the composition was photographed initially, shaken for 6.5 days in an orbital mixer set to "5" on a scale of 0 to 10, and then photographed again. The initial and aged compositions were visually compared, and differences were determined. All test compositions showed turbidity after 6.5 days, indicating that the surfactant blend in the test composition was able to overcome the incompatibility between each individual surfactant and bacterial spores.

[0083] Example 9: Testing of sodium lauryl sulfate, lauramine oxide, and surfactant blends Three test compositions were prepared: one containing an aqueous solution of the anionic surfactant sodium lauryl sulfate (STEPANOL® WA-Extra HP), another containing an aqueous solution of the lauramine oxide surfactant (AMMONYX® LO from Stepan Company), and a third containing an aqueous solution of a blend of these two surfactants. Bacterial spores were added to each of the three test compositions to evaluate the ability of the surfactant blends to overcome the antagonistic incompatibility between each surfactant individually and the bacterial spores. Each test composition was prepared by adding 1 × 10⁶ units of water.7 The test compositions contained a commercially available Bacillus spore blend at CFU / g and 0.2% by weight of surfactants as active ingredients. Each test composition containing the surfactant blend contained 0.1% by weight of each surfactant as an active ingredient, for a total of 0.2% by weight of surfactants as active ingredients. Each test composition also contained 2% by weight of a 3.3 g / L nutritional mix (3 g / L of tripty soy broth and 0.3 g / L of yeast extract), and the pH of the test compositions was adjusted to pH 7 using L-lactic acid or dilute NaOH as needed.

[0084] The test compositions were visually evaluated for bacterial spore germination and growth using the unstained EVOO test described above. However, the test compositions were photographed after being shaken for 5 days in an orbital mixer set to setting "5".

[0085] Figure 11 is a photograph showing the jars of the test compositions. The jar on the left contains a test composition containing only sodium lauryl sulfate, the jar in the center contains a test composition containing only lauramine oxide, and the jar on the right contains a test composition containing a blend of the two surfactants. As shown in the photograph, the test composition containing the blend of the surfactants sodium lauryl sulfate and lauramine oxide had significant turbidity due to biological growth in the test composition, while the test composition containing the surfactants individually showed strong inhibition of Bacillus species, was much clearer, and had the same visual turbidity as the first Bacillus population alone. The blend of these two surfactants can overcome the inhibitory effect on Bacillus species that each surfactant would exhibit if not blended.

[0086] Example 10: Structured surfactant system containing anionic and nonionic surfactants Aqueous test compositions containing blends of incompatible low-HLB nonionic surfactants and incompatible high-HLB surfactants were prepared to evaluate the ability of surfactant blends to form structured aqueous systems capable of suspending bacterial spores. The test compositions contained glyceryl caprylate / caprate mono- and di-esters (STEPANMILD® GCC) ("GCC") as the low-HLB surfactant and alpha-sulfonated alkyl ester (ALPHA-STEP PC-48) ("PC-48") as the anionic high-HLB surfactant. Each test composition contained GCC and PC-48 at a total active ingredient concentration of approximately 15.5% by weight, with the relative active ingredient weight ratio of these two surfactants varying from 1:3 to 3:1. Each test composition also contained 1% by weight of alkyl sarcosinate surfactant (Maprosyl® 30-B) (0.3% active ingredient), 0.2% by weight of 2-phenoxyethanol, and 2.68 × 10⁻⁶ 9 The mixture also contained a commercially available Bacillus spore blend at CFU / ml, and water in an amount that made up 100% by weight of the composition.

[0087] The test compositions were prepared by first combining GCC and PC-48 in water, and then by secondly adding alkyl sarcosinate, Bacillus spores, 2-phenoxyethanol, and water. The test compositions were visually inspected for their ability to form a structured surfactant system capable of suspending bacterial spores. The formation of a structured surfactant system can be visually determined by observing the presence of suspended bubbles throughout the system, an increase in viscosity, and stability as a homogeneous solution. From the tests, it was found that active ingredient weight ratios of GCC to PC-48 within the range of approximately 2:1 to 2.5:1 were able to form a structured system capable of suspending Bacillus spores, while an active ingredient weight ratio of 3:1 resulted in immediate separation.

[0088] To evaluate the effect of changes in spore concentration on the stability of the formulation, a test composition containing an active ingredient weight ratio of approximately 2.3:1 for GCC to PC-48 was selected. The test composition contained a total active ingredient concentration of approximately 15.5% by weight of GCC and PC-48, 1% by weight of alkyl sarcosinate (0.30% active ingredient), 0.2% by weight of 2-phenoxyethanol, and water, but the spore concentration was varied. The spore concentration tested was 1.0 × 10⁻⁶. 11 The test compositions contained 0%, 1.8%, 2.5%, and 5% by weight of commercially available Bacillus spore blends with CFU / ml spores. The active ingredient weight ratio of GCC to total anionic surfactant (PC-48 + alkyl sarcosinate) in the compositions was approximately 2.2:1. The test compositions containing 0%, 1.8%, and 2.5% by weight of the Bacillus spore blend formed a stable structured surfactant system that suspended the spores. The test composition containing 5% by weight of the Bacillus spore blend failed to maintain a stable suspension and exhibited layer separation.

[0089] Example 11: Rheological properties of a spore-containing structured system Test compositions from Example 10, having a GCC to PC-48 active ingredient weight ratio of approximately 2.3:1 and containing spore concentrations of 0 wt% spores and 2.5 wt% Bacillus spore blends, were tested to determine the effect of bacterial spores on the rheological properties of the compositions. The viscosity of the test compositions as a function of shear rate was measured using a rheometer at 25°C. The graph in Figure 12 shows the results of the viscosity tests. As shown in Figure 12, the test compositions containing 2.5 wt% Bacillus spore blend exhibited dramatically lower viscosity compared to the spore-free test compositions across the entire shear rate range from 0.1 / sec to 100 / sec. However, in the spore-containing compositions, the structuring achieved by the surfactant combination still maintained a relatively high viscosity (approximately 10,000 cP) at low shear rates. Under high shear, the viscosity decreased to approximately 100 cP, making the composition easier to handle. The spore-free test compositions exhibited relatively high viscosity even under high shear, indicating that they were more difficult to handle than the spore-containing compositions.

[0090] The viscosity of the test compositions was also measured as a function of temperature, by varying the temperature from 5°C to 60°C at a fixed shear rate of 60 / second. Both test compositions (with and without spores) showed fairly consistent viscosity over this temperature range, particularly at temperatures above 20°C; however, overall, the viscosity of the spore-containing compositions was lower than that of the spore-free compositions.

[0091] Example 12: Compatibility of structured surfactant blends and spores Aqueous test compositions were prepared from test formulations containing glyceryl caprylate / caprate mono- and di-esters (STEPANMILD® GCC) ("GCC") and alpha-sulfonated alkyl ester (ALPHA-STEP PC-48) ("PC-48") to evaluate the ability of the surfactant blend to overcome antagonistic incompatibility between each surfactant individually and bacterial spores. The test formulations contained GCC and PC-48 at a total active ingredient concentration of approximately 15.5% by weight and an active ingredient weight ratio of approximately 2.3:1 for GCC to PC-48. The test formulations also contained 1% by weight of an alkyl sarcosinate surfactant (Maprosyl® 30-B) (0.3% active ingredient), 0.2% by weight of 2-phenoxyethanol, and 2.68 × 10⁻⁶ 9 The mixture also contained a commercially available Bacillus spore blend at CFU / ml, and water in an amount totaling 100% by weight of the composition. The weight ratio of GCC to total anionic surfactant (PC-48 + alkyl sarcosinate) was approximately 2.2:1.

[0092] Repeat test samples were prepared by diluting the test formulation with water to a dilution ratio of 0.3% by weight in a test jar, and adding 1% by weight of a 33 g / L nutritional mix (30 g of tripty soy broth and 3.0 g / L of yeast extract). The test samples were evaluated for bacterial germination and growth using the Sudan III staining EVOO test described in Example 7, however, instead of using an orbital mixer, the test samples were placed on a stirrer plate with a stirrer bar in each jar and stirred at a speed of 500 rpm. After 6 days of mixing, the test samples were photographed and showed significant turbidity, indicating that the surfactant blend in the formulation was able to overcome the incompatibility between the individual surfactants and bacterial spores.

[0093] Example 13: Structured surfactant system including surfactant blend as an alternative option Aqueous test compositions containing different combinations of unsuitable low-HLB and high-HLB surfactants were prepared to determine the ability of these surfactant combinations to form a structured surfactant system capable of suspending spores. The surfactant combinations tested were ethoxylated alcohol (BIOSOFT® N91-2.5) as the low-HLB surfactant and alpha-sulfonated methyl ester (ALPHA-STEP® PC-48), alkyl ether sulfate (STEOL® CS-270C or CS-270 PLUS), or alkylamine oxide (AMMONYX® LO) as the high-HLB surfactant. Each test composition contained a combination of the low-HLB and high-HLB surfactants at a total active ingredient concentration of approximately 16% by weight, with the relative weight ratio of these two surfactants varying from 14:7 to 7:14. Each test composition also contained 1% by weight of an alkyl sarcosinate surfactant (Maprosyl® 30-B) (0.3% by weight of active ingredient), 0.25% by weight of 2-phenoxyethanol, and water in an amount totaling 100% by weight of the composition. The tests showed that combining ethoxylated alcohols and amine oxides in a product weight ratio of ethoxylated alcohol to amine oxide within the range of approximately 14:7 to 10:13 could form a structured surfactant system capable of suspending spores. Combinations of ethoxylated alcohols with alpha-sulfonated alkyl esters and combinations of ethoxylated alcohols with alkyl ether sulfates showed phase separation without forming a structured surfactant system.

[0094] Test compositions containing surfactant combinations of ethoxylated alcohol and amine oxide in product weight ratios of 13.5:8, 12:10, and 10:13 were evaluated for yield stress, which is determined from amplitude sweeps measuring the storage modulus and loss modulus as functions of amplitude stress. In this embodiment, yield stress is the viscous transfer stress value at which the loss modulus is higher than the storage modulus. The yield stress values ​​of the test compositions with surfactant weight ratios of 13.5:8 and 12:10 were in the range of approximately 130 to approximately 170 Pa, while the yield stress value of the composition with a surfactant weight ratio of 10:13 was approximately 50 Pa. These results indicate that the compositions with surfactant weight ratios of 13.5:8 and 12:10 can provide yield stress values ​​that are sufficient to suspend spores.

[0095] The effect of spore addition on the yield strength of the composition was determined by adding spores of different concentrations to test compositions containing ethoxylated alcohol and amine oxide in weight ratios of 13.5:8 and 12:10. The tested spore concentrations were 100 × 10⁻⁶. 9 The test results were obtained using commercially available Bacillus spore blends containing spores at concentrations of 0%, 1.8%, and 2.5% by weight. The test results are shown in the graph in Figure 13. The yield stress graph in Figure 13 shows that the yield stress of the composition decreases significantly when spores are added to it. These results also show that a weight ratio of ethoxylated alcohol to amine oxide of 12:10 yields higher values ​​compared to a ratio of 13.5:8, and that at a weight ratio of 12:10, a spore concentration of 2.5% resulted in a higher yield stress than a concentration of 1.8%.

[0096] Test compositions with surfactant weight ratios of 13.5:8 and 12:10, and spore concentrations of 1.8% by weight and 2.5% by weight, were visually evaluated for stability at two different temperatures: 25°C and 50°C. At 25°C, the composition with a surfactant ratio of 12:10 provided a structured surfactant system that stably suspended spores at both concentrations of 1.8% by weight and 2.5% by weight, while the composition with a ratio of 13.5:8 could only stably suspend spores at a concentration of 1.8% by weight. At 50°C, the composition with a weight ratio of 13.5:8 was unstable and separated at both spore concentrations, while the composition with a weight ratio of 12:10 was still able to suspend spores.

[0097] Example 14: Ethoxylated alcohol / amine oxide surfactant blend and spore compatibility Aqueous test compositions were prepared from test formulations containing surfactant blends of ethoxylated alcohol and alkylamine oxide from Example 13 to evaluate the ability of the surfactant blends to overcome antagonistic incompatibility between each surfactant individually and bacterial spores. The test formulations contained the surfactant blend at a product weight ratio of approximately 12:10 for ethoxylated alcohol to amine oxide, a weight ratio of approximately 4.2:1 for ethoxylated alcohol to amine oxide active ingredient, and a total active ingredient concentration of approximately 16% by weight. The test formulations also contained 1% by weight of alkyl sarcosinate surfactant (Maprosyl® 30-B) (0.3% active ingredient), 0.2% by weight of 2-phenoxyethanol, and 2.55 × 10⁻⁶ 9 The mixture also contained a commercially available Bacillus spore blend at CFU / ml, and water in an amount totaling 100% by weight of the composition. The weight ratio of the anionic surfactant (alkyl sarcosinate) to the amphoteric surfactant (alkylamine oxide) was approximately 0.1:1.

[0098] The test compositions were prepared in two separate batches at two different concentrations by diluting the test formulation with water to 0.2% and 1.0% by weight in test jars, and adding 1% by weight of 33 g / L nutritional mix (30 g of tripty soy broth and 3.0 g / L of yeast extract) to each test composition. The test compositions were evaluated for bacterial germination and growth using the Sudan III staining EVOO test described in Example 7. The test compositions were photographed initially and again after 6 days of mixing. Both concentrations of the test compositions showed significant turbidity, indicating that the surfactant blend in the formulation was able to overcome the incompatibility between the individual surfactants and bacterial spores.

[0099] Example 15: Diesel fuel digestion In this example, the ability of Bacillus spores to germinate, grow, and digest diesel fuel when present in an aqueous composition containing an incompatible surfactant blend was investigated. Repeat test samples were prepared by combining 48.3 g of DI water, 1.0 g of the formulation from Table 3 prepared with dialkylamide, 1.0 g of sodium xylenesulfonate (80% active ingredient), 0.2 g of a 33 g / L nutrient mix, and 0.5 g of a stock suspension of a commercially available Bacillus spore mix at 10^9 CFU / g. 2.0 g of Sudan III-stained (62.5 ppm) diesel fuel was added to the test samples. Repeat control samples were prepared similarly, except that Sudan III-stained (62.5 ppm) EVOO was added to the control samples. The test and control samples were mixed on the same stirring plate at 500 rpm for 15-30 minutes, phase separation was achieved, and then photographs were taken. The samples were then stirred for 7 days to allow for phase separation, and photographed again. The test samples showed significant turbidity similar to that of the EVOO control samples. These results indicate that the Bacillus spore mix in the test samples was able to germinate, grow, and digest diesel fuel.

[0100] Example 16: Preservative experiment Preservative screening experiments were conducted to identify the amount of preservatives in selected MEC formulations sufficient to provide efficacy against pathogenic bacteria and fungi. The base formulations used in these experiments were similar to composition A in Example 5, but without spores, and similar to the formulation in Example 14, but without spores and 2-phenoxyethanol. Test formulations were prepared by adding different amounts of different preservatives to the base formulations. Each test formulation was tested for efficacy against pathogenic organisms using a preservative screening test. In the screening test, microorganisms were grown separately according to the industry standard described in PCPC Test Method M-7 "A Rapid Method for Preservation Testing of Water-Miscible Personal Care Products," combined as an inoculant, and then introduced into the formulation test sample. The inoculant was added to each test formulation at predetermined time points, and after appropriate nutritional supplementation, the test formulation was evaluated for residual viable cell count. The reduction in viable cell count was calculated relative to the control, and samples showing a sufficient reduction rate were accepted as effectively preserved formulations. Table 4 shows the details of the preservatives used in each test formulation and their quantities, as well as the results of the screening tests.

[0101] [Table 4]

[0102] These results indicate that, in either base formulation, 1.5% by weight of 2-phenoxyethanol is insufficient to provide efficacy against pathogenic bacteria and fungi, even at pH 9.0. However, both base formulations passed the screening test with a combination of 1.5% by weight (0.75% by weight each) of 2-phenoxyethanol and benzoic acid. These results also indicate that base formulations containing other preservatives known in the art passed the screening test for efficacy against pathogenic bacteria and fungi.

[0103] Further embodiments of the present invention are described in the following numbered paragraphs. Paragraph 1. A composition for overcoming antagonistic surfactant incompatibility between surfactants and beneficial microorganisms, comprising: about 2.0% to about 15% by weight of sodium lauryl ether sulfate; 0% to about 2.0% by weight of alcohol alkoxylate; about 2.0% to about 15% by weight of cocoamidopropyl betaine; 0% to about 2% by weight of dimethyl lauramide / myristamide or lauryl lactyl lactate; 0% to about 7.0% by weight of MgCl2 or NaCl; at least 10 4 A composition comprising beneficial live microorganisms in CFU / mL and water in an amount totaling 100% by weight of the composition.

[0104] Paragraph 2. The composition according to Paragraph 1, wherein the alcohol alkoxylate is present in the composition in an amount of 0.1% to 2% by weight of the active ingredient, and comprises a linear alcohol ethoxylate.

[0105] Paragraph 3. The composition according to Paragraph 2, wherein the linear alcohol ethoxylate has 8 moles of ethoxylate per molecule. Paragraph 4. A composition for overcoming antagonistic surfactant incompatibility between surfactants and beneficial microorganisms, the composition comprising: sodium lauryl sulfate in an amount of about 0.1% to about 15% by weight of active ingredient; lauramine oxide in an amount of about 0.1% to about 15% by weight of active ingredient; and at least 10 4 A composition comprising beneficial live microorganisms in CFU / mL and water in an amount totaling 100% by weight of the composition.

[0106] Paragraph 5. A composition according to any one of paragraphs 1 to 4, further comprising one or more enzymes, preferably a protease, amylase, lipase, or a combination thereof. Paragraph 6. A method for overcoming antagonistic surfactant incompatibility between surfactants and beneficial living microorganisms, the method comprising (a) at least 10 4(b) a step of providing beneficial live microorganisms in cfu / mL; (c) a step of providing an anionic surfactant in an amount of 0.1% to 15% by weight of active ingredients based on the total weight of the composition, which alone exhibits antagonistic surfactant incompatibility when in contact with beneficial live microorganisms and adversely affects the viability, growth, or metabolic activity of beneficial live microorganisms, wherein the anionic surfactant is selected from the group consisting of alkyl sulfates, alkyl ether sulfates, alkyl sarcosinates, alpha-sulfonated alkyl esters, alkyl glutamates, and combinations thereof; (d) a step of providing a further surfactant in an amount of 0.1% to 15% by weight of active ingredients, which alone exhibits antagonistic surfactant incompatibility when in contact with beneficial live microorganisms and adversely affects the viability, growth, or metabolic activity of beneficial live microorganisms, wherein the further surfactant active ingredients are (i) alkylamine oxides, alkyl betaines, alkylamidopropyl betaines A method comprising: (ii) an amphoteric surfactant selected from the group consisting of (ii) alkyl sulfobetaine and combinations thereof, and (ii) a nonionic surfactant selected from the group consisting of glyceryl monoester and / or glyceryl diester; (d) optionally providing a nonionic surfactant other than glyceryl monoester and glyceryl diester that, when alone, exhibits antagonistic surfactant incompatibility when in contact with beneficial living microorganisms and adversely affects the viability, growth, or metabolic activity of beneficial living microorganisms; and (e) mixing beneficial microorganisms, anionic surfactant, further surfactant, and optionally nonionic surfactant together in a liquid carrier to form a composition, wherein the anionic surfactant, further surfactant, and optionally nonionic surfactant, when combined, overcome antagonistic surfactant incompatibility between the surfactant and beneficial living microorganisms and promote the viability, growth, and metabolic activity of beneficial living microorganisms.

[0107] Paragraph 7. The method according to Paragraph 6, wherein the anionic surfactant is an alkyl ether sulfate in an amount of 2.0% to 15% by weight of the active ingredient, the amphoteric surfactant is an alkylamidopropyl betaine in an amount of 2.0% to 15% by weight of the active ingredient, and the nonionic surfactant is an alcohol ethoxylate in an amount of 0.1% to 2% by weight of the active ingredient.

[0108] Paragraph 8. The method according to paragraph 6 or paragraph 7, further comprising mixing a dialkylamide or alkyl lactyl lactate in an amount of 0.1% to 2% by weight of the active ingredient with an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant.

[0109] Paragraph 9. The method according to any one of paragraphs 6 to 8, further comprising mixing 0.1% to 7.0% by weight of MgCl2 or NaCl with an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant.

[0110] Paragraph 10. The method according to Paragraph 6, wherein the anionic surfactant is a combination of an alpha-sulfonated alkyl ester in an amount of about 2% to about 7% by weight of the active ingredient and an alkyl sarcosinate in an amount of about 0.2% to about 2% by weight of the active ingredient, and the further surfactant is a glyceryl monoester and / or glyceryl diester in an amount of about 5% to about 15% by weight of the active ingredient.

[0111] Paragraph 11. The method according to Paragraph 6, wherein the anionic surfactant is an alkyl sarcosinate in an amount of about 0.2% to about 2% by weight of the active ingredient, the amphoteric surfactant is an alkylamine oxide in an amount of 2% to about 5% by weight of the active ingredient, and the nonionic surfactant is an ethoxylated alcohol with an HLB value of less than 10 in an amount of about 8% to about 20% by weight, or as an alternative, about 8% to about 15% by weight of the active ingredient.

[0112] Paragraph 12. An embodiment according to any one of paragraphs 1 to 11, wherein the composition maintains beneficial microorganisms in a suspended state without separation for at least one month. This document describes the technology in sufficient, clear, and concise language so that those skilled in the art in which the technology pertains can put it into practice. It should be understood that the foregoing describes preferred embodiments of the technology, and that modifications may be made without departing from the spirit or scope of the technology as set forth in the appended claims. Furthermore, the examples are provided not exhaustively, but to illustrate some embodiments that fall within the scope of the claims. Any references cited in the detailed description section of this application are incorporated herein by reference in their entirety unless otherwise noted.

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