Novel protective barrier composition and its use

Abstract

The present invention provides compositions and methods for enhancing the barrier function of surfaces and / or objects using biochemical-producing microorganisms and / or by-products synthesized by such microorganisms. [Solution] A composition is provided comprising a Lactobacillus species grown as a biofilm and / or an extract of the biofilm, wherein when the composition is applied to a surface, the biofilm and / or extract enhance the barrier function such that the surface is less susceptible to the effects of microorganisms, viruses, and / or chemical contamination.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 940,598, filed on November 26, 2019, and U.S. Provisional Patent Application No. 63 / 016,336, filed on April 28, 2020, each of which is incorporated herein by reference in its entirety.

[0002] Sequence List The sequence list of this application is named "SeqList - 24Nov20_ST25.txt", which was created on November 24, 2020, and is 2 KB. The sequence list is incorporated herein by reference in its entirety.

Background Art

[0003] Background of the Invention Chemical and biological threats are dangerous for both military and civilian people around the world. Chemical threats can include blister agents and nerve agents that often cause long - term effects or death. Biological threats can include bacteria, viruses, and fungi that are dispersed as a means of harming other organisms such as humans or crops. Common examples of current biological threats include Bacillus anthracis and plague.

[0004] To limit or prevent the effects of harmful chemical and biological threats, appropriate protective or countermeasures are essential. Barriers can be established to address many chemical and biological threats.

[0005] Barriers can prevent the intrusion or direct contact of threats to humans. Barriers can be used to neutralize, isolate, or otherwise inactivate threats. Barriers can prevent the adhesion of threats or enhance the dispersion of threats. With respect to biological threats, barriers can actively prevent or stop means of growth, including inhibiting biofilm formation or spore formation.

[0006] Like many biological threats, the bacterium Yersinia pestis, the causative agent of the plague, relies on its ability to form biofilms. The efficient transmission of Y. pestis cells depends on biofilm formation in the foregut of fleas, which inhibits the digestion of blood meal and subsequently facilitates the reflux of blood meal containing Y. pestis cells into humans.

[0007] Attacking, dissolving, or otherwise weakening bacterial biofilm matrices can be helpful in dealing with biological pathogens, respectively. Controlling pathogenic bacteria, biofilms, and other substances on the skin or in other organs is also a key element in restoring or maintaining the health of soldiers and civilians. However, current treatments focus primarily on symptom control rather than modulating the overall microbiome to inhibit the proliferation of chemical or biological threats and reduce their adhesion and attachment.

[0008] New tools are needed to combat the growing populations of harmful organisms and substances. Preventing contact or adhesion of harmful organisms or substances, neutralizing or isolating harmful organisms or substances, and improving barrier durability are all methods that can work collectively or independently to promote improvements in abiotic and biotic barrier functions. [Overview of the project]

[0009] The present invention provides compositions and methods for creating or enhancing biological and / or non-living barriers. A preferred embodiment provides biofilm-producing bacteria and / or bioactive substances synthesized by such bacteria, preferably when grown in a biofilm phenotype.

[0010] In a preferred embodiment, the composition of the present invention comprises secretions from a biofilm. In one embodiment, the secretions can be purified from the culture before being added to the composition. The present invention also provides compositions comprising, for example, lyophilized and / or freeze-dried cell lysates.

[0011] In some embodiments, the bacterial strain is Lactobacillus fermentum Qi6, also referred to herein as Lf Qi6. In one embodiment, the present invention provides isolated or biologically pure cultures of Lf Qi6. In another embodiment, the present invention provides biologically pure cultures of Lf Qi6 grown as a biofilm. Biofilm phenotypes, as well as methods utilizing extracts of biofilm phenotypes and their lysates, are also provided herein.

[0012] In certain embodiments, natural barrier function is enhanced by modifying barriers in the lungs, eyes, nose, mouth, trachea, esophagus, ears, stomach, intestines, and / or brain.

[0013] In one aspect, the present invention preferably has one or more biological activities selected from inhibiting antimicrobial activity, inhibiting the growth of pathogenic biofilms, inhibiting the adhesion of pathogenic biofilms, promoting the detachment of pathogenic biofilms, promoting the growth of symbiotic biofilms, and enhancing skin barrier function.

[0014] In a particular aspect, the present invention provides a self-contamination-free surface.

[0015] In an exemplary embodiment, the pathogenic bacterium is methicillin-resistant Staphylococcus aureus (MRSA), and the symbiotic bacterium is Staphylococcus epidermidis (S. epidermidis).

[0016] In certain embodiments, the composition can improve the durability of existing barriers or create new, durable barriers and / or be self-decontaminating. In preferred embodiments, the barrier is stable and retains its ability to decontaminate chemical and / or microbial contamination for at least 1, 2, 3, 7, 10, 30, or 180 days or longer.

[0017] In certain embodiments, the composition creates or enhances a barrier on inanimate objects and / or surfaces. The objects may be large surfaces such as ship hulls and airplane wings, or fine surfaces such as instruments used to repair nerves. The compositions and methods of the present invention can prevent contamination of inanimate objects, and in the case of surgical instruments, can prevent bacterial infection. Preferably, the compositions and methods of the present invention prevent or limit direct contact of living or non-living entities on the object, isolate living or non-living entities, and / or inactivate living or non-living entities. In this respect, the surface may be self-decontaminating.

[0018] In a preferred embodiment, the present invention provides a bioactive protein having barrier functional properties. In a particular embodiment, the present invention provides "Qi611S," a protein having an amino acid sequence by SEQ ID NO. 1. In a particular embodiment, the present invention also provides "Qi611S protein," which includes Qi611S and its bioactive fragments and variants. [Brief explanation of the drawing]

[0019] [Figure 1] The inhibition of MRSA biofilm adhesion at 3 × 10⁸ CFU / ml upon exposure to 32 mg / ml of Qi601SL, Qi601SP, Qi601SML, Qi601SMP, or Qi medium as a negative control is illustrated. Cultures were washed twice with deionized water. [Figure 2]Inhibition of MRSA biofilm adhesion at 3×108 CFU / ml when exposed to 8 mg / ml, 16 mg / ml, or 32 mg / ml of Qi601SL or Qi601SP in water or PBS is illustrated. The cultures were not washed with deionized water. [Figure 3] Inhibition of MRSA biofilm adhesion at 3×108 CFU / ml when exposed to 32 mg / ml of Qi601SL, Qi601SP, Qi601SML, Qi601SMP, or Qi medium as a negative control is illustrated. The cultures were washed three times with deionized water. [Figure 4] Inhibition of MRSA biofilm adhesion at 3×108 CFU / ml when exposed to a solution of 3.2% Qi601S is illustrated. The cultures were washed once, twice, three times, or four times with deionized water. [Figure 5] Inhibition of MRSA biofilm adhesion at 3×108 CFU / ml when exposed to serial dilution solutions of Qi601SL or Qi601SP containing 3.2%, 1.6%, 0.8%, 0.4%, 0.2%, 0.1%, 0.05%, and 0.025% is illustrated. The cultures were washed three times with deionized water. [Figure 6] Inhibition of MRSA biofilm adhesion at 3×108 CFU / ml when exposed to serial dilution solutions of Qi601SL or Qi601SML containing 3.2%, 1.6%, 0.8%, 0.4%, 0.2%, 0.1%, 0.05%, and 0.025% is illustrated. The cultures were not washed with deionized water. [Figure 7] Inhibition of MRSA biofilm adhesion at 1.5×106 CFU / ml when exposed to Qi601AM is illustrated. The cultures were washed once, twice, three times, or zero times with deionized water. [Figure 8] Inhibition of MRSA biofilm adhesion at 1.5×106 CFU / ml when exposed to Qi601A is illustrated. The cultures were washed once, twice, three times, or zero times with deionized water. [Figure 9] An atomic force microscope image of the composition of the present invention. [Figure 10]Qi 601S is shown to create an anti-adhesive surface effective against Escherichia coli (E. coli) on Caco-2 cells. Cells were split into 24-well plates at 5×104 cells per well and grown for an additional 10 days for differentiation and confluence density. Selected wells were pretreated overnight with 0.1% v / v Qi 601S and then washed with PBS. Escherichia coli K12 was grown overnight in TSB, 1×108 CFU was added to each well, and incubated for 3 hours. Unbound bacteria were removed by PBS washing. Intestinal cells were lysed with 1% Triton X-100. Bound bacteria were quantified by serial dilution and standard plate count in TSA and compared to untreated controls. [Figure 11] An anti-adhesive surface against MRSA produced by Qi 601S is shown. 1% v / v Qi 601S in PBS was applied to living human skin during culture for 24 hours and then rinsed. The skin was then cultured with MRSA for 2 days, the skin was rinsed, and then MRSA colonies were cultured. Pretreatment with Qi 601S demonstrated a significant reduction in MRSA load of over 60% compared to untreated skin. [Figure 12] A hydrophilic plot of Qi611S. [Figure 13] Hydrophilic thermostable QI fraction Qi 601S shows AChE protection. Qi601S administered at 2% provided 100% protection of AChE activity with all doses of donepezil used. **BEST MODE FOR CARRYING OUT THE INVENTION**

[0020] **BRIEF DESCRIPTION OF THE SEQUENCES** SEQ ID NO: 1 is the amino acid sequence of a protein named "Qi611S". SEQ ID NO: 2 is the nucleotide sequence Qi611S encoding the amino acid sequence of SEQ ID NO: 1.

[0021] **DETAILED DESCRIPTION OF THE INVENTION** The present invention provides compositions and methods for creating or enhancing barriers on living or non-living surfaces. Preferred embodiments of the present invention provide compositions comprising Lactobacillus species and / or bioactive extracts thereof that can grow in a biofilm phenotype, and methods for using the same. The present invention also provides compositions of Lactobacillus species and / or bioactive extracts thereof in lyophilized, freeze-dried, and / or lysated forms.

[0022] Advantageously, the preferred compositions and treatment methods provided herein are effective in strengthening existing barriers or creating new barriers in both living and non-living materials. Living organisms and materials treated with this approach may exhibit reduced fouling, particularly by bacterial biofilms.

[0023] In certain embodiments, the present invention provides a self-decontamination surface. For example, in a preferred embodiment, the composition may be self-decontamination by preventing or reducing the adhesion of microorganisms, viruses, and / or chemicals to a surface, such as a surface containing animal tissue cells. In a preferred embodiment, this barrier function is independent of antigens and pathogens.

[0024] In some embodiments, the Lactobacillus species is Lactobacillus fermentum Qi6, referred to herein as Lf Qi6. In one embodiment, the method utilizes an extract of the biofilm phenotype, including the biofilm phenotype and a lysate of the biofilm phenotype. In a preferred embodiment, the composition comprises a bioactive extract of the Lf Qi6 biofilm.

[0025] In a preferred embodiment, the present invention provides a bioactive protein having barrier functional properties. In a particular embodiment, the present invention provides "Qi611S," a protein having an amino acid sequence by SEQ ID NO. 1. In a particular embodiment, the present invention also provides "Qi611S protein," which includes Qi611S and its active fragments and variants.

[0026] In some embodiments, the Qi611S protein can be produced by cells, preferably bacterial cells. Therefore, in certain embodiments, the present invention provides a method for producing the Qi611S protein, comprising the step of culturing cells having a nucleotide sequence encoding all or part of SEQ ID NO. 1, or a variant or fragment thereof, under conditions favorable for the expression of the protein. In a preferred embodiment, the nucleotide sequence is Qi611s (SEQ ID NO. 2). Optionally, the protein can be purified from the culture.

[0027] In one embodiment, the method utilizes a microorganism having the Qi611s nucleotide sequence (SEQ ID NO. 2), such as Lactobacillus fermentum Qi6. Qi611s encodes the amino acid sequence by SEQ ID NO. 1 (Qi611S).

[0028] In another embodiment, the cells are a microorganism that has been recombinantly modified to possess the ability to express the Qi611S protein. In a particular embodiment, the microorganism possesses all or part of the Qi611s gene. Thus, in a particular embodiment, the present invention provides recombinant cells that can express a protein having all or part of the DNA sequence according to SEQ ID NO. 2 and / or the amino acid sequence according to SEQ ID NO. 1, or a fragment or variant of SEQ ID NO. 1. In an exemplary embodiment, the recombinant cells are Escherichia coli BL21 or Escherichia coli C43.

[0029] Such transformation of cells can be achieved using techniques well known to those skilled in the field of microbiology. In one embodiment, the nucleotide sequence can be modified to optimize the expression of the Qi611S protein.

[0030] In a preferred embodiment, the present invention provides a composition comprising cells containing all or part of the Qi611S protein and / or the DNA sequence according to SEQ ID NO. 2, and optionally a carrier. Cultures of the L. fermentum microorganism are deposited at the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 USA. The depositary was assigned accession number ATCC No. PTA-122195 by the depositary and was deposited on June 10, 2015.

[0031] The culture is deposited under the condition that it be made available during the pendency of this patent application to any person whom the Commissioner of the Patent and Trademark Office deems eligible to acquire it, under 37 CFR 1.14 and 35 USC 122. The deposited material may be made available in accordance with the requirements of the foreign patent law of the country in which a copy of this application or its products is filed. However, it should be understood that the use of the deposited material does not constitute authorization to practice the invention in a manner that restricts the application of any patent rights granted by administrative measures.

[0032] Furthermore, the culture deposit shall be stored and made publicly available in accordance with the provisions of the Budapest Convention on the Deposit of Microorganisms, that is, it shall be stored with all necessary care to keep it alive without contamination for at least five years from the most recent request for distribution of the deposited sample, and in any case for at least 30 years from the date of deposit, or for the duration of any patent that may be exercised to make the disclosure of the culture public. The depositor accepts the obligation to replace the deposited sample if the storage institution is unable to distribute the sample at the time of request due to the condition of the deposited sample. All restrictions on the public availability of the culture deposit shall be irrevocably removed upon the granting of a patent disclosing it.

[0033] Selected definition "Qi611S" refers to the protein having the amino acid sequence SEQ ID NO: 1. A singular or plural reference to "Qi611S protein" refers to Qi611S, as well as its active fragments and variants.

[0034] As used herein, “gene” refers to a segment or nucleotide sequence of DNA capable of expressing polypeptides and / or amino acid chains. In certain embodiments, the gene includes regions such as a promoter region before and / or after a coding region.

[0035] As used herein, “biologically pure culture” means a culture isolated from other bioactive substances, including any substances that may have been associated with it in nature. In a preferred embodiment, the culture is isolated from all other living cells. In a further preferred embodiment, the biologically pure culture has advantageous characteristics compared to cultures of the same microbial species that may exist in nature. These advantageous characteristics may be, for example, enhanced production of one or more desirable growth byproducts.

[0036] In a particular embodiment, the purified compound is at least 60% by weight of the compound of interest. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest. For example, the purified compound is preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the desired compound by weight. Purity is measured by any suitable standard method, for example, by column chromatography, thin-layer chromatography, or high-performance liquid chromatography (HPLC) analysis.

[0037] As used herein, the term “subject” refers to an animal that needs or desires the delivery of benefits provided by an active agent (e.g., a pharmaceutical compound). The animal may be, for example, a human, a pig, a horse, a goat, a cat, a mouse, a rat, a dog, a great ape, a fish, a chimpanzee, an orangutan, a guinea pig, a hamster, a cattle, a sheep, a bird (including a chicken), and any other vertebrate or invertebrate. These benefits may include, but are not limited to, the treatment of a health condition, disease, or disorder; the prevention of a health condition, disease, or disorder; the promotion of immune health; and / or the enhancement of the function of an organ, tissue, or system within the body. A preferred subject in the context of the present invention is a human. In some embodiments, the subject suffers from a health condition, disease, or disorder; on the other hand, in some embodiments, the subject is in good health (e.g., free from injury or illness) but desires the enhancement of the health and / or function of a particular organ, tissue, or system of the body. The target audience may be any age or developmental stage, including infants, toddlers, adolescents, teenagers, adults, or older adults.

[0038] As used herein, the terms “therapeutic effective dose,” “therapeutic effective dosage,” “effective dose,” and “effective dosage” are used to mean the amount or dosage of a compound or composition that, when administered to a subject, can treat or improve a condition, disease, or disorder in the subject, or provide an enhancement of health or function to an organ, tissue, or system of body. In other words, the amount administered to a subject is the “therapeutic effective dose.” The actual amount will vary depending on many factors, including but not limited to the specific condition, disease, or disorder being treated or improved; the severity of the condition; the specific organ, tissue, or system of body for which health or function enhancement is desired; the patient’s weight, height, age, and health status; and the route of administration. Prescribing treatment, such as determining the dosage, is the responsibility of the general practitioner and other physicians, and typically takes into account the disorder to be treated, the individual patient’s condition, the site of administration, the method of administration, and other factors known to the practitioner.

[0039] As used herein, the term “treatment” means eradicating, reducing, relieving, or reversing to any degree the signs or symptoms of a health condition, disease, or disorder, including, but not required to, a complete cure of the condition, disease, or disorder. Treatment may cure, improve, or partially relieve a disorder. Treatment may also include improving or enhancing a condition or characteristic, such as bringing the function of a particular system in the body to a healthy or homeostatic state.

[0040] As used herein, “preventing” a health condition, disease, or disorder means avoiding, delaying, preventing, or minimizing the onset of any particular sign or symptom of that condition, disease, or disorder. Prevention may be absolute or complete, but does not have to be; it means that there is still a possibility that the sign or symptom may occur thereafter. Prevention may include reducing the severity of the onset of such condition, disease, or disorder, and / or preventing the condition, disease, or disorder from progressing to a more severe condition, disease, or disorder.

[0041] As used herein, a “microbe-based composition” or “composition of a microbial source” means a composition containing components produced as a result of the growth of a microorganism or other cell culture. A microbe-based composition may contain the microorganism itself; or the microorganism may be isolated from the broth or medium in which it was cultured, and therefore the composition may contain residual cellular components and / or by-products of microbial growth. By-products of microbial growth may be, for example, metabolites, cell membrane components, synthesized proteins, and / or other cellular components.

[0042] The present invention further provides “microbe-based products” which are products to be actually applied to achieve desired results. A microbe-based product may simply be a microbe-based composition recovered from a microbial culture process. Alternatively, a microbe-based product may include further added components. These additional components may include, for example, stabilizers, buffers, and / or suitable carriers (e.g., water or saline solutions). A microbe-based product may include a mixture of microbe-based compositions. A microbe-based product may also include one or more components of a microbe-based composition processed by any means, such as filtration, centrifugation, dissolution, drying, and purification.

[0043] As used herein, “recovered” refers to removing some or all of the microbial-based composition from the growth vessel.

[0044] As used herein, “applying” a composition or product means bringing it into contact with a target or site so that the composition or product may have an effect on that target or site. This effect may, for example, result from the growth of microorganisms and / or the action of their growth byproducts. For example, a microorganism-based composition or product may be sprayed onto an object and / or surface.

[0045] As used herein, “isolated” or “purified” proteins are substantially free from other compounds, such as cellular material, that it associates with in nature or within growth vessels. In certain embodiments, the purified compound is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest. For example, the purified compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (w / w) of the desired compound by weight. Purity is measured by any suitable standard method, for example, by column chromatography, thin-layer chromatography, or high-performance liquid chromatography (HPLC) analysis.

[0046] A "metabolite" refers to any substance produced by metabolism (i.e., a growth by-product) or a substance required to participate in a particular metabolic process. Metabolites can be organic compounds that are metabolic starting materials (e.g., glucose), intermediates (e.g., acetyl-CoA), or final products (e.g., n-butanol). Examples of metabolites include, but are not limited to, biosurfactants, enzymes, acids, solvents, gases, alcohols, proteins, vitamins, minerals, trace elements, amino acids, and polymers.

[0047] The term "adjust" refers to a change (increase or decrease). Such changes are detected by standard methods known in the art, such as those described herein.

[0048] The ranges provided herein are understood to be all abbreviated representations of the values ​​within that range. For example, the range 1–20 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, as well as all decimal values ​​interposing between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either endpoint of a range are specifically intended. For example, nested subranges of the exemplary range 1–50 may include 1–10, 1–20, 1–30, and 1–40 in one direction, or 50–40, 50–30, 50–20, and 50–10 in the other direction.

[0049] "Decrease" means a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0050] "Increase" means a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0051] "Reference" means a standard or contrasting state.

[0052] As used herein, “pharmaceutical,” “health-promoting compound,” or “health-promoting substance” refers to a compound manufactured for use as a pharmaceutical and / or therapeutic agent.

[0053] As used herein, “contaminant” means any substance that contaminates or defiles another substance or object. A contaminant may be living or non-living, and may be inorganic or organic, a substance or deposit. In one embodiment, a contaminant is a virus. Living organisms may include bacteria, such as cyanobacteria, species of the genera Pseudomonas, Bacillus, Listeria, Staphylococcus, Lactobacillus, and Lactococcus, and eukaryotes, such as algae, yeasts, fungi, barnacles, and mussels. Furthermore, contaminants may include, but are not limited to, scale, hydrocarbons such as petroleum, tar sands, or asphaltenes; fats, oils, and greases (FOGs) such as cooking grease and lard; lipids; waxes such as paraffin; resins; biofilms; or any other substance referred to as, for example, mud, dust, sludge, sediment, tailings, grime, floating debris, plaque, deposits, or residues. References to “scale” mean, for example, any kind of scale resulting from the precipitation of barium sulfate, calcium carbonate, calcium sulfate, calcium oxalate, magnesium hydroxide, magnesium oxide, silicates, strontium sulfate, aluminum hydroxide oxide, aluminosilicates, magnetite or nickel ferrite, sodium chloride, silicon dioxide, iron sulfide, iron oxide, iron carbonate, copper, phosphates, oxides, and any other inorganic compounds that may precipitate and form deposits.

[0054] The harmful accumulation of substances, whether living or non-living, leads to a process of "fouling." Fouling can result in clogging, grime formation, or other undesirable buildup. Fouling can affect the efficiency, reliability, or functionality of an object.

[0055] The transitional term “comprising” is synonymous with “including” or “containing,” and is comprehensive or non-restrictive, not excluding additional elements or method steps that are not listed. In contrast, the transitional phrase “consisting of” excludes any elements, steps, or components that are not specified in the claims. The transitional phrase “essentially consisting of” limits the scope of the claims to the specified material or steps and those that “do not substantially affect the basic and novel features” of the claimed invention, such as the ability to inhibit bacterial growth. The use of the term “consisting” is intended to describe the modes of “consisting of” and “essentially consisting of” the listed components.

[0056] Unless specifically stated or evident from the context, the term “or” is understood to be inclusive as used herein. Unless specifically stated or evident from the context, the terms “a,” “an,” and “it” are understood to be singular or plural as used herein.

[0057] Unless otherwise specifically stated or made clear from the context, the term “about” as used herein is understood to mean within the range of normal tolerances in the art, for example, within two standard deviations of the mean. The term “about” may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0058] As used herein, “biofilm” refers to a complex aggregate of microorganisms, such as bacteria, in which cells are typically adhered to one another by a matrix composed of, but not limited to, polysaccharide materials. Cells in a biofilm are physiologically different from planktonic cells of the same organism, which are single cells that can float or swim in a liquid or gaseous medium, or reside on or within a solid or semi-solid surface. Individual microbial cells can also be fibrous, linked together in a chain-like manner, without forming a distinct biofilm. However, the fibrous nature of cells can facilitate biofilm formation.

[0059] Any enumeration of chemical groups in any definition of a variable herein includes the definition of that variable as any single group or combination of the enumerated groups. Any enumeration of embodiments of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or part thereof. Any composition or method provided herein may be combined with any or more other compositions and methods provided herein.

[0060] Other features and advantages of the present invention will become apparent from the following description of its preferred embodiments and from the claims. All references cited herein are incorporated herein by reference.

[0061] Lactobacillus species and Qi601S lysate Lactobacillus species are Gram-positive rods. Lactobacillus fermentum Qi6 (Lf Qi6) can be grown in MRS medium at 37°C. Lf Qi6 cultures can be used in the present disclosure, or bioactive lysates can be isolated from Lf Qi6 cultures and used in the antimicrobial and other barrier enhancement / creation methods and compositions of the present invention.

[0062] In addition, Lf Qi6 can be grown in a biofilm phenotype. Lf Qi6 cultures can be used in accordance with this disclosure, or bioactive lysates, including Qi601S, Qi601SM, Qi601SP, Qi601SL, Qi601SMP, Qi601SML, Qi601ML, Qi601MP, Qi601AM, and Qi601A, can be isolated from Lf Qi6 biofilm cultures and used in antimicrobial or other barrier enhancement / creation methods and compositions. Qi601 represents a bioactive lysate of Lf Qi6, and each letter accompanying Qi601 represents a different fraction isolated from the culture, or a method of growing and / or processing the culture. Qi601S, Qi601SM, Qi601A, and Qi601AM are different fractions isolated from Lf Qi6. "L" and "P" represent the liquid and powder / lyophilized preparations of the fraction, respectively.

[0063] In one method for growing a culture to form a biofilm, the culture can be incubated in 5 ml of MRS broth at 37°C for 24 hours. Then, 1 ml of the culture can be transferred to a T-150 tissue culture plate containing 25 ml of MRS broth. Next, 25 ml of MRS medium can be replaced every 48 hours to allow the Lf Qi6 biofilm to grow as a flora on the bottom of the culture plate. The culture can then be grown for, for example, 7 days to produce a thick biofilm layer. After that, the grown biofilm layer can be peeled off and suspended in fresh medium. Freezer storage can be prepared using glycerol and stored at -80°C.

[0064] The biofilm phenotype of Lf Qi6 from frozen storage can be cultured in 10 ml of fresh MRS medium at 37°C for 24 hours. 10 ml of the culture can be inoculated into 25 L of MRS medium containing 500 g of sterile glass wool. The biofilm can then be cultured under static conditions at 37°C for 72 hours. The culture can be mixed every 24 hours by gentle shaking, after which the medium and glass wool can be collected. Subsequently, the biofilm cells can be separated from the glass wool by sonication. The cells can be further centrifuged to concentrate the Lf Qi6 biofilm, which is then suspended in sterile water. This scale-up yields a biofilm culture with a concentration of 2 g / L.

[0065] Polynucleotide sequences encoding the Qi611S protein and the 611S protein. In a preferred embodiment, the present invention provides proteins useful for creating barriers, as well as fragments and variants thereof. The present invention further provides nucleotide sequences encoding such proteins, as well as fragments and variants thereof.

[0066] In a particular embodiment, the protein of the present invention, referred to as "Qi611S," has a molecular weight of approximately 8.0 kDA. The "Qi611S protein," including Qi611S and its fragments and variants, can be characterized according to several parameters, including, for example, the ability to create or contribute to a barrier that protects a surface from contamination by microorganisms, viruses, and / or chemicals.

[0067] The Qi611S protein can be further defined by its amino acid sequence. In a particular embodiment (Qi611S), the protein has a 74-amino acid sequence indicated as SEQ ID NO: 1.

[0068] In a particular embodiment, the proteins provided herein may also be identified based on their immunoreactivity with a particular antibody.

[0069] In a particular embodiment, the Qi611S protein is produced by the Lactobacillus fermentum Qi6 bacterial strain when laboratory growth conditions are used to induce a biofilm phenotype during growth. In a preferred embodiment, this bacterial strain has the Qi611S DNA sequence (SEQ ID NO: 2), which can express a protein having SEQ ID NO: 1 under biofilm phenotypic conditions.

[0070] Lactobacillus fermentum is a Gram-positive rod-shaped bacterium. Lactobacillus fermentum Qi6 (Lf Qi6) can be grown in MRS medium at 37°C.

[0071] In a particular embodiment, the polynucleotide sequence is Qi611S, which is 222 base pairs and encodes Qi611S; however, in a particular embodiment, for example due to genetic code redundancy, a different DNA sequence may encode the amino acid sequence disclosed herein. It is well within the art of the art to create these alternative DNA sequences that encode the Qi611S protein.

[0072] As used herein, a "variant" of a protein refers to a sequence having one or more amino acid substitutions, deletions, additions, or insertions. In a preferred embodiment, these substitutions, deletions, additions, or insertions do not substantially adversely affect the barrier activity of Qi611S. Variants that retain barrier activity are within the scope of the present invention.

[0073] Fragments of Qi611S and its variants are also within the range of the Qi611S protein, insofar as the fragment retains one or more biological properties of Qi611S. Preferably, one or more biological activities include barrier-promoting activity. Preferably, the fragment is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of full-length Qi611S. The fragment may, for example, comprise one or more hydrophilic domains of Qi611S or its variants. These domains may be directly linked with intervening amino acids removed. The hydrophilic domains can be readily identified using standard procedures known in the art and as illustrated in Figure 12.

[0074] The present invention further envisions a fusion construct in which the Qi611S protein is directly or indirectly (e.g., via a linker) bound to another portion that may be, for example, a targeting site (e.g., a ligand, antibody, or aptamer), a toxin, a carrier, a label, or an activity enhancer.

[0075] The present invention further envisions antibodies against the Qi611S protein (e.g., polyclonal, monoclonal, chimeric, and humanized). These antibodies can be readily prepared by those skilled in the art having the teachings provided herein. These antibodies can be used, for example, in protein purification.

[0076] In certain embodiments, the polynucleotide encoding the Qi611S protein can be isolated, amplified, and ligated into a vector. A “vector,” “plasmid,” or “plasmid vector” is a DNA molecule used to transfer DNA into a cell, often from one cell to another (a host cell). A vector can replicate within a host cell; or a vector can be a means of incorporating DNA into a cell (or removing DNA from a cell). Vectors can be introduced into host cells by various means. Some cells can take up vectors without any action by those skilled in the art other than introducing the vector into the cell culture. Others require chemical modification. Regardless of the means by which a cell can take up a vector, once a host cell has the ability to do so, it is now a “competent” cell.

[0077] In a particular embodiment, the present invention relates to gene transformation of host cells to provide host cells with the ability to produce the Qi611S protein. For example, a vector having Qi611S (or other polynucleotides encoding the Qi611S protein) can be used to transform host cells (e.g., microorganisms, plants, fungi, and / or animal cells) to enable the use of recombinant cells for the production of the Qi611S protein.

[0078] In a preferred embodiment, the host cell is a strain of Escherichia coli, for example, E. coli BL21 or E. coli C43. Alternatively, the ability to transform non-E. coli cells into competent cells is well understood in the art, and this includes cells selected based on, for example, their transformative ability, ability and efficiency of heterologous protein expression, protein stability in the host, presence of accessory gene capacity, lack of mammalian toxicity, ease of killing and fixing without damaging proteins, ease of culturing and / or formulation, ease of handling, cost-effectiveness, storage stability, and similar factors.

[0079] It will be recognized by those skilled in the art that the DNA sequences of the present invention can be varied by the degeneracy of the genetic code and the use of codons. All DNA sequences encoding the Qi611S protein are intended. Therefore, all polynucleotide sequences encoding the Qi611S protein, including the DNA encoding SEQ ID NO: 1 (optionally including ATG before the coding region), are included in the present invention. The present invention also includes polynucleotides having codons optimized for expression in host cells, including any of the specific types of cells mentioned herein. Various techniques for producing optimized sequences are known in the art.

[0080] Furthermore, it will be recognized by those skilled in the art that allelic mutations can occur in a DNA sequence that do not significantly alter the activity of the amino acid sequence of the peptide encoded by the DNA sequence. All such variant DNA sequences are included within the scope of the present invention.

[0081] Those skilled in the art will understand that, using the exemplified sequences, additional nucleotide sequences encoding the Qi611S protein can be identified, generated, and used. Variant DNA sequences having at least 90% or at least 95% identity with the enumerated DNA sequences and encoding the Qi611S protein are included in the present invention. Other numerical ranges for variant polynucleotides and amino acid sequences are provided below (e.g., 50–99%). Following the teachings herein and using knowledge and techniques well known in the art, those skilled in the art will be able to create numerous effective embodiments having variant DNA sequences without using excessive experimentation. Homologous forms from other strains or species are specifically intended.

[0082] The polynucleotide and amino acid sequence fragments, as well as mutations, insertions, and deletion variants of the present invention, can be used in the same manner as the exemplified sequences, provided that the fragments and variants have substantial sequence similarity to the original sequence. As used herein, substantial sequence similarity refers to the degree of nucleotide or amino acid sequence similarity sufficient to enable the variant or fragment sequence to function with the same capabilities as the original sequence. Preferably, this similarity is greater than 50%; more preferably, greater than 75%; and most preferably, greater than 90%. The degree of similarity required for a variant to function with its intended capabilities depends on the intended use of the sequence. It is within the scope of the art to create mutations, insertions, and deletion variants designed to improve the function of a sequence or otherwise provide methodological advantages. Identity and / or similarity may also be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% compared to the sequences exemplified herein.

[0083] The identity / similarity and / or homology of amino acids is typically highest in critical regions of a protein that are responsible for biological activity and / or involved in determining the three-dimensional arrangement that ultimately carries that activity. In this regard, certain amino acid substitutions may be acceptable and predictable if they are in regions that are not critical to activity or are conserved amino acid substitutions that do not affect the three-dimensional arrangement of the molecule. For example, amino acids can be classified into the following classes: nonpolar, non-charged, basic, and acidic. Conservative substitutions in which an amino acid of one class is replaced by another amino acid of the same type fall within the scope of this invention, provided that the substitution does not substantially alter the biological activity of the compound. Below (Table 1) is a list of examples of amino acids belonging to each class.

[0084] (Table 1) Classification of amino acids based on physical properties TIFF2026062963000002.tif42153

[0085] In some cases, non-conservative substitutions can be created. The important factor is that these substitutions must not significantly impair the biological activity of the protein.

[0086] Formulation and delivery of Lf Qi6 and Qi601S dissolved products The present invention provides compositions and methods for enhancing barrier function. A preferred embodiment of the present invention provides compositions comprising novel solubilants and methods for using them. The present invention also provides compositions in lyophilized and / or freeze-dried forms.

[0087] Advantageously, preferred compositions and processing methods provided herein are effective in treating or preventing bacterial and / or viral infections, preventing contamination, limiting or eliminating contact between objects and harmful substances or organisms, inactivating harmful organisms or substances, and / or increasing the durability of barriers.

[0088] In a particular aspect, the present invention provides a self-contamination-free surface.

[0089] In one aspect, the present invention provides compositions for preventing or mitigating chemical or microbial contamination of surfaces and / or for treating bacterial and / or viral infections, comprising a biologically pure bacterial strain and / or its bioactive extract, and one or more pharmaceutically acceptable excipients. In addition, the compositions may consist of bacterial strains capable of growing in both planktonic and biofilm phenotypes, and the compositions may have one or more bioactivities selected from general antimicrobial activity, inhibition of pathogenic biofilm growth, inhibition of pathogenic biofilm adhesion, promotion of pathogenic biofilm detachment, promotion of symbiotic biofilm growth, and enhancement of skin barrier function.

[0090] In some embodiments, self-decontamination surfaces prevent the adhesion of microorganisms or viruses while enhancing the barrier function of the substrate involved, and in certain embodiments, prevent subsequent biofilm formation and / or detach existing biofilms. In certain embodiments, the compositions comprise one or more thermostable biofilm proteins that, when applied to surfaces such as skin, as well as biological surfaces such as polystyrene and glass, inhibit the formation of harmful biofilms and / or other contaminants. These compositions improve skin immunity, physical cohesiveness, and the overall barrier function of the skin. These compositions provide alternatives to conventional chemical surface decontaminations and pharmacological antimicrobial agents. Because they are non-toxic to human biological surfaces, their biomedical applications are broad, including use as self-decontamination substances applied topically to the skin, intestines, stomach, lungs, eyes, mouth, sinuses, nose, ears, trachea, or esophagus of the subject.

[0091] As used herein, the term “extract” refers to a composition obtained by processing a bacterial culture. Processing may include, for example, physical and / or chemical treatments. Physical and / or chemical treatments may include, for example, filtration, centrifugation, sonication, pressurization, radiation, dissolution, treatment with a solvent or other chemical, and combinations thereof. The extract may be in the form of a supernatant, for example, produced by centrifugation. The extract may also include cell aggregates obtained by centrifugation. The cells may be intact or not, and may be viable or not. The extract may include cell membrane components and / or intracellular components. In certain embodiments, the extract is at least 80, 85, 90, or 95% by weight of cell aggregates. In certain embodiments, at least 95% of the intact cells are not viable. In certain embodiments, less than 10% of the cell aggregates in the extract are intact cells.

[0092] Human skin consists of two compartments: the deep compartment (dermis) and the superficial compartment (epidermis). The skin constitutes a barrier against external attacks, specifically chemical, mechanical, or infectious attacks, as well as several defensive responses against environmental factors such as climate, ultraviolet radiation, and tobacco, and / or xenobiotic factors such as microorganisms. This property is called the skin barrier function and is primarily provided by the outermost layer of the epidermis, i.e., the stratum corneum. Harmful changes in the barrier can be reflected, for example, by skin discomfort, sensory phenomena, and / or dryness.

[0093] Compositions according to some aspects of the present invention are useful for preventing a decline in barrier function and / or for repairing or regenerating barrier function. Disorders associated with the breakdown of the skin and / or mucous membrane barrier include, but are not limited to, psoriasis, ichthyosis, sarcoidosis, atherosclerosis, inflammatory bowel disease, acne (including hidradenitis suppurativa), burns, diaper dermatitis, Netherton syndrome, actinic keratosis, cutaneous mycosis, dermatopathies or ectodermal dysplasia, atopic dermatitis, contact dermatitis, seborrheic dermatitis, plaque, eosinophilic esophagitis, filaggrin deficiency, and other disorders associated with damage or breakdown of the skin and / or mucous membrane barrier.

[0094] In some embodiments, the methods of the present invention promote the repair, regeneration, or other strengthening of barriers, including the repair or regeneration of mucous membranes. Mucous membranes include oral mucosa (including the mucosa of the cheeks, soft palate, tongue including the underside of the tongue, and floor of the mouth), nasal mucosa, pharyngeal mucosa (including the mucosa of the pharynx, larynx, trachea, and esophagus), bronchial mucosa, pulmonary mucosa, ocular mucosa, otorhinolary mucosa, gastrointestinal mucosa, vaginal mucosa, penile mucosa, urethral mucosa, bladder mucosa, and anal mucosa. In certain embodiments, compositions of the present invention may also be used to treat acute and chronic viral infections. In particular, the present invention may be used to treat chronic Epstein-Barr virus infection, influenza infection, coronavirus (including COVID-19) infection, cytomegalovirus infection, and other herpes-type viral infections, which are ubiquitous in populations and associated with reduced immune surveillance, or to create barriers against their entry. Some viral infections can cause cancer. For example, Epstein-Barr virus infection can be a risk factor for Hodgkin lymphoma, a cancer of lymphocytes.

[0095] Advantageously, preferred compositions of the present invention can be heat-sterilized and retain their activity. In one embodiment, compositions of the present invention retain their ability to resist microbial colonization on surfaces even after being subjected to 254°F and a pressure of 21 pounds / square inch for 30 minutes. In preferred embodiments, activity is maintained at pH 4.0–8.0, 4.5–7.5, or 5.0–7.5.

[0096] The pharmaceutical compositions provided herein also include, but are not limited to, pharmaceutically acceptable carriers, auxiliaries, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, and colorants, and may also include other pharmaceutically acceptable components known to those skilled in the art. The formulations may further include other active agents, for example, other therapeutic or prophylactic agents.

[0097] Where provided herein, “pharmaceutically acceptable” means that it is approved or may be approved by a U.S. federal or state regulatory authority, or that it is listed in the United States Pharmacopeia or any other commonly recognized pharmacopoeia for use in animals, including humans.

[0098] "Pharmacologically acceptable excipients, carriers, or adjuvants" means excipients, carriers, or adjuvants that can be administered to a subject together with the active ingredient and that do not impair its pharmacological activity and are nontoxic when administered in a dose sufficient to deliver a therapeutic dose of the composition provided herein.

[0099] Infections occur when disease-causing microorganisms invade the tissues of the body. Such microorganisms and the toxins they produce often react with the body's tissues, triggering an immune response by the infected host. Infections can be caused by bacteria, viruses, viroids, fungi, and other parasites. Infections can occur through any of the body's tissues, such as the skin, intestines, or membranes. In certain embodiments, the present invention provides compositions for treating and / or preventing infections of the body's external surface, and specifically the skin. Infections can be caused by bacteria, such as pathogenic Staphylococcus aureus. The pharmaceutical compositions provided herein may be applied separately, sequentially, or simultaneously with exposure to infectious agents. In other embodiments, the present invention provides materials and methods for treating intestinal disorders and other internal disorders.

[0100] Staphylococcus aureus (S. aureus) is a transient colonizing bacterium of the skin, primarily found in moist, warm areas of the body, such as the groin, armpits, and anterior nostrils. Up to 60% of the world's population are intermittent carriers, while another 20% may have a stable colony. While typical carriers are asymptomatic, Staphylococcus aureus can invade tissues (e.g., through damaged skin), where it can cause diseases ranging from relatively mild impetigo and scalded skin syndrome to life-threatening conditions such as sepsis. Furthermore, Staphylococcus aureus infections are often secondary to underlying conditions such as atopic dermatitis (AD).

[0101] Exemplary compositions provided by the present invention include species of the genus Staphylococcus (Staphylococcus saprophyticus, Staphylococcus xylosus, Staphylococcus lugdunensis, Staphylococcus schleiferi, Staphylococcus caprae, Staphylococcus saprophyticus, Staphylococcus hominis, Staphylococcus aureus), species of Pseudomonas, Enterococcus faecalis, vancomycin-resistant enterococci (VRE), Bacillus cereus, Bacillus subtilis, and Listeria monocytogenes. It is useful for treating infections caused by several pathogenic bacteria, including but not limited to Listeria monocytogenes, Streptococcus pyrogenes, Streptococcus salivariu, Streptococcus mutans, and Streptococcus pneumoniae, or as a barrier against them. Other pathogenic bacteria will be readily recognized by those skilled in the art.

[0102] In certain embodiments, the present invention provides antimicrobial compositions in the form of cleaning products, cleaning solutions, surface coatings, or other compositions, not for medical treatment of human or animal bodies. Accordingly, in certain embodiments, these compositions are used to disinfect inanimate surfaces or to provide a barrier against microbial colonization of inanimate surfaces. In certain embodiments, the present invention provides self-contamination-free surfaces.

[0103] In another aspect, the present invention provides a method for treating a human dermatological disorder or providing a barrier against such disorder, comprising the step of administering an effective amount of a composition to a target, wherein the composition preferably comprises one or more bioactive extracts of Lf Qi6 biofilm.

[0104] The compositions may be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the treatment or condition to be protected. The compositions provided herein may be dissolved in, suspended in, or mixed with one or more other acceptable components. The compositions may also be present in liposomes or other microparticles.

[0105] In a preferred embodiment, the composition is formulated for topical administration, specifically for use or application to or on the skin. Such formulations may be useful for removing, killing, or preventing the adhesion, establishment, and / or accumulation of undesirable substances, such as pathogenic bacteria like MRSA, on biological or abiotic surfaces, or for inhibiting the action or growth of bacteria. Furthermore, in a particular embodiment, a composition comprising a biofilm of Lf Qi6 or an extract thereof has the advantage of promoting the growth of symbiotic bacteria in the human skin microbiome. Non-exclusive examples of symbiotic bacteria include, but are not limited to, Staphylococcus epidermidis, Staphylococcus warneri, Streptococcus mitis, Propionibacterium acnes, Corynebacterium species, Acinetobacter johnsonii, and Pseudomonas aeruginosa.

[0106] The compositions provided herein may preferably be provided as patches, bandages, dressings, or similar items impregnated with or coated with extracts of a microbial biofilm and / or one or more thereof, and optionally with one or more other acceptable components, such as penetration, permeation, and absorption enhancers.

[0107] Compositions according to aspects of the present invention may be useful for treating biomaterials, implants, and orthotics (including stents, valves, eyes, hearing aids, gastric bands, dentures, and artificial joint replacements), surgical instruments, or other medical devices prior to administration to a subject, treatment of a subject, or use on a subject. Antimicrobial compositions may be useful for treating surfaces that are prone to bacterial or viral colonization or exposure, such as handrails, food preparation surfaces, kitchen surfaces or equipment, tables, sinks, toilets, or other bathroom equipment.

[0108] The composition may include, in addition to microbial (e.g., Lf Qi6) biofilms or their bioactive extracts, agents such as detergents, stabilizers, anionic surfactants, fragrances, chelating agents, acids, alkalis, buffers, and / or detergents. Such agents may promote or enhance the antimicrobial and / or barrier properties of the composition, such as by killing or inhibiting bacteria and / or viruses or preventing their adhesion to surfaces.

[0109] Formulations suitable for skin and / or transdermal administration include, but are not limited to, gels, pastes, ointments, creams, lotions, and oils, as well as patches, bandages, dressings, depots, cements, glues, and reservoirs.

[0110] The ointment is typically prepared from the cosmetic compositions provided herein and a paraffinic or water-miscible ointment base.

[0111] The cream is typically prepared from the cosmetic compositions and oil-in-water cream bases provided herein. Optionally, the aqueous phase of the cream base may contain, for example, at least about 30% w / w of polyhydric alcohols, i.e., alcohols having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol, polyethylene glycol, and mixtures thereof.

[0112] As will be readily apparent to those skilled in the art, the formulations according to the present invention may also contain other alcohols, such as isopropyl alcohol or ethanol, and may cover other alcohol-based formulations, such as alcohol-based hand sanitizers.

[0113] Topical formulations may preferably contain compounds that enhance the absorption or penetration of the active compound through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogues.

[0114] The emulsion is typically prepared from the cosmetic compositions and oil phases provided herein, and may optionally contain only an emulsifier (otherwise known as an emulsion), or it may contain a mixture of at least one emulsifier with fat or oil, or both fat and oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier acting as a stabilizer. It is also preferable to include both oil and fat. Together, the emulsifier, with or without a stabilizer, constitutes a so-called emulsifying wax, and the wax, together with the oil and / or fat, constitutes a so-called emulsifying ointment base that forms the oily dispersion phase of the cream formulation.

[0115] Suitable emulsions and emulsifying stabilizers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate. Since the solubility of active compounds in most oils likely to be used in pharmaceutical emulsion formulations is considered very low, the selection of an oil or fat suitable for the formulation is based on achieving the desired cosmetic properties. Therefore, the cream should preferably be a non-greasy, non-staining, and washable product with an appropriate consistency to avoid leakage from tubes or other containers. Linear or branched monobasic or dibasic alkyl esters, such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a mixture of branched esters known as Crodamol CAP, may be used, with the latter three being preferred esters. These can be used alone or in combination, depending on the required properties. Alternatively, high-melting-point lipids such as white soft paraffin and / or liquid paraffin, or other mineral oils may be used. Other formulations include dental sprays, mouthwashes, toothpastes, lozenges, antimicrobial cleaning solutions, beverages (e.g., milk, yogurt), foods (yogurt, ice cream, candy bars, etc.), or powdered foods (e.g., powdered milk).

[0116] The compositions provided herein may comprise a single dose of probiotic bacteria or their lysates or extracts. A suitable dose of probiotic bacteria (intact, lysed, or extracted) may be within the range of 10⁴–10¹² cfu, e.g., 10⁴–10¹⁰, 10⁴–10⁸, 10⁶–10¹², 10⁶–10¹⁰, or 10⁶–10⁸⁰ cfu. In some embodiments, the dose may be administered once or twice daily. In some embodiments, the composition for use according to the present invention may contain, by weight, at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, or about 6.0%. It may contain extracts of approximately 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 25.0%, 30.0%, 35.0%, 40.0%, 45.0%, and 50.0%. In some embodiments, the composition may contain at least one Lf Qi6 extract by weight in the following proportions: about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 5%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, and about 1% to about 5%.

[0117] For the purposes of this invention, the abbreviation cfu specifies a “colony-forming unit” which is defined as the number of bacterial cells indicated by the number of microorganisms on an agar plate.

[0118] In one embodiment, the composition is formulated as an orally consumable product such as a food, capsule, tablet, or drinking liquid. Orally deliverable health-promoting compounds are any bioactive substances delivered via initial absorption in the gastrointestinal tract or into the oral mucosa. The composition can also be formulated as a solution that can be administered by injection, for example, intravenous, intraperitoneal, intramuscular, intrathecal, or subcutaneous. In another embodiment, the composition is formulated to be administered via the skin through a patch or directly to the skin for topical or systemic effects. The composition can also be administered sublingually, buccally, rectally, or vaginally. Furthermore, the composition can be sprayed into the nose for absorption through the nasal membrane, nebulized, inhaled through the mouth or nose, or administered into the eyes or ears.

[0119] An orally consumable product according to the present invention is any preparation or composition suitable for consumption, nutrition, oral hygiene, or palatability, which is intended to be introduced into the oral cavity of a human or animal, remain there for a period of time, and then be swallowed (e.g., ready-to-consume food or tablets) or removed from the oral cavity again (e.g., chewing gum or oral hygiene product or medical mouthwash). Orally deliverable pharmaceuticals can be formulated into orally consumable products, and orally consumable products may include orally deliverable pharmaceuticals, but the two terms are not intended to be used interchangeably herein.

[0120] Orally consumable products include all substances or products intended to be ingested by humans or animals in a processed, semi-processed, or unprocessed state. This also includes substances added to orally consumable products (especially food and pharmaceutical products) during their manufacture, processing, or modification and intended to be introduced into the oral cavity of humans or animals.

[0121] Orally consumable products may also contain substances intended to be swallowed by humans or animals and subsequently digested in an unmodified, prepared, or processed state. Orally consumable products according to the present invention may also include casings, coatings, or other encapsulations intended to be swallowed or expected to be swallowed with the product.

[0122] In one embodiment, the orally consumable product is a capsule, pill, syrup, emulsion, or liquid suspension containing the desired orally deliverable substance. In one embodiment, the orally consumable product may include an orally deliverable substance in powder form that can be mixed with water or another liquid to produce an orally consumable product suitable for drinking.

[0123] In some embodiments, the orally consumable products according to the present invention may comprise one or more formulations intended for nutrition or palatability. These include baking products (e.g., bread, dried biscuits, cakes, and other pastries), confectionery (e.g., chocolate, chocolate bar products, other bar products, fruit gum, coated tablets, hard caramel, toffee and caramel, and chewing gum), alcoholic or non-alcoholic beverages (e.g., cocoa, coffee, green tea, black tea, black tea or green tea beverages fortified with green tea or black tea extract, rooibos tea, other herbal teas, fruit-containing lemonade, isotonic beverages, soft drinks, nectar, fruit and vegetable juices, and fruit or vegetable juice preparations), instant beverages (e.g., instant cocoa beverages, instant tea beverages, and instant coffee beverages), meat products (e.g., ham, fresh or raw sausage preparations, and seasoned or marinated raw or salted meat products), eggs or egg products (e.g., dried whole eggs, egg whites, and egg yolks), cereal products (e.g., breakfast cereals, muesli bars, and cooked instant rice products), and dairy products (e.g., whole fats). This includes low-fat or non-fat milk beverages, rice pudding, yogurt, kefir, cream cheese, soft cheese, hard cheese, dried milk powder, whey, butter, buttermilk, and partially or completely hydrolyzed products containing milk protein), soy protein or other products derived from soy fractions (for example, soy milk and its preparations, beverages containing isolated or enzymatically treated soy protein, soy flour beverages, preparations containing soy lecithin, fermented products such as tofu or tempeh preparations, and fruit preparations and optionally flavorings). Mixtures of substances), fruit preparations (e.g., jams, fruit ice creams, fruit sauces, and fruit fillings), vegetable preparations (e.g., ketchup, sauces, dried vegetables, flash-frozen vegetables, cooked vegetables, and boiled vegetables), snacks (e.g., baked or fried potato chips (crisps), or potato dough products, and corn or peanut-based extrudes), oils and fats or their emulsion-based products (e.g., mayonnaise, remoulade, and dressings), other ready-made meals and soups (e.g.,This includes dried soups, instant soups, and prepared soups, seasonings (e.g., sprinkle seasonings), and sweetener compositions (e.g., tablets, packets, and other preparations for sweetening or whitening beverages or other foods). This composition may also function as a semi-finished product for the production of other compositions intended for nutrition or flavor.

[0124] The composition may further comprise one or more pharmaceutically acceptable carriers and / or excipients and can be formulated in preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

[0125] The carriers and / or excipients according to the present invention include any solvent, diluent, buffer (neutral buffered saline, phosphate-buffered saline, or optionally Tris-HCl, acetic acid, or phosphate buffer), oil-in-water or water-in-oil emulsions, aqueous compositions with or without organic cosolvents suitable for use IV, solubilizers (e.g., Polysorbate 65, Polysorbate 80), colloids, dispersion media, media, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, fragrances, thickeners (e.g., carbomer, gelatin, or sodium alginate), coating agents, preservatives (e.g., thimerosal, benzyl alcohol, polyquatherium). This may include (polyquaterium), antioxidants (e.g., ascorbic acid, sodium metabisulfite), osmotic regulators, absorption retarders, adjuvants, bulking agents (e.g., lactose, mannitol), and similar substances. The use of carriers and / or excipients in the field of drugs and supplements is well known. Except for any conventional media or active substances that are incompatible with the target health-promoting substance or adjuvant composition, the use of carriers or excipients in this composition may be contemplated.

[0126] In one embodiment, the composition may be made into an aerosol formulation so that it can be sprayed or inhaled, for example, by a nebulizer. Pharmaceutical formulations suitable for administration in the form of an aerosol or spray are, for example, liquids, suspensions, or emulsions. Formulations for oral or nasal aerosol or inhalation administration may also be formulated using exemplary carriers, for example, physiological saline, polyethylene glycol or glycol, DPPC, methylcellulose, or mixed with powder dispersants or fluorocarbons. Aerosol formulations may be placed in pressurized sprays such as dichlorodifluoromethane, propane, nitrogen, fluorocarbons, and / or other solubilizers or dispersants known in the art. Exemplarily, delivery may be by the use of a single-use delivery device, a mist nebulizer, a respiratory-operated powder inhaler, an aerosol metered-dose inhaler (MDI), or any other of the many nebulizer delivery devices available in the art. In addition, direct administration via a mist tent or endotracheal tube may be used.

[0127] In one embodiment, the composition may be formulated for administration by injection, for example, as a liquid or suspension. The liquid or suspension may contain a suitable non-toxic, parenterally acceptable diluent or solvent, such as mannitol, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution, or a suitable dispersant or wetting agent and suspension agent, such as a sterile, non-irritating fixative oil containing synthetic monoglycerides or diglycerides, as well as fatty acids containing oleic acid. One exemplary example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600, and the remainder USP Water for Injection (WFI). Other exemplary carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01–0.1% triethanolamine in USP WFI; or 0.01–0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1–10% squalene or parenteral aqueous vegetable oil emulsion. Water or saline solution, as well as aqueous dextrose and glycerol solutions, may be used as carriers, preferably especially for injectable solutions. Examples of carriers for subcutaneous or intramuscular use include phosphate-buffered saline (PBS) solution, 5% dextrose in WFI and 0.01–0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1:2 or 1:4 mixture of 10% USP ethanol and 40% propylene glycol, and an acceptable isotonic solution of the remainder such as 5% dextrose or 0.9% sodium chloride; or 0.01–0.2% dipalmitoyl diphosphatidylcholine and 1–10% squalene or a vegetable oil-in-water emulsion in USP WFI.

[0128] In one embodiment, the composition can be formulated for administration via topical application to the skin, for example, as a topical solution, which includes rinses, sprays, drops, lotions, gels, ointments, creams, foams, powders, solids, sponges, tapes, vapors, pastes, tinctures, or transdermal patches. A suitable formulation for topical application may contain, in addition to any of the pharmaceutically active carriers, emollients such as carnauba wax, cetyl alcohol, cetyl ester wax, emulsifying wax, hydrated lanolin, lanolin, lanolin alcohol, microcrystalline wax, paraffin, petrolatum, polyethylene glycol, stearic acid, stearyl alcohol, white beeswax, or yellow beeswax. In addition, the composition may contain humectants such as glycerin, propylene glycol, polyethylene glycol, sorbitol solution, and 1,2,6-hexanetriol, or permeation enhancers such as ethanol, isopropyl alcohol, or oleic acid.

[0129] One bacterial-based product of the present invention is simply a growth medium containing bacteria and / or microbial metabolites and / or any residual nutrients produced by the bacteria. The growth product can be used directly without extraction or purification. If desired, extraction and purification can be easily achieved using standard extraction and / or purification methods or techniques described in the literature.

[0130] The bacteria in the bacteria-based product may be in an active or inactive form. The bacteria-based product can be used without further stabilization, preservation, or storage. Advantageously, the direct use of these bacteria-based products maintains a high viability of the microorganisms, reduces the possibility of contamination from foreign matter or undesirable microorganisms, and preserves the activity of the by-products of microbial growth.

[0131] In one embodiment, the lysate may be a purified form or a mixture of the growth product. The lysate can be added at a concentration of 0.01 to 90 wt%, preferably 0.1 to 50 wt%, and more preferably 0.1 to 20 wt%. In another embodiment, the lysate can be combined with an acceptable carrier, in which case the lysate may be expressed at a concentration of 0.001 to 50% (v / v), preferably 0.01 to 20% (v / v), and more preferably 0.02 to 5% (v / v).

[0132] Useful surfactants and solvents according to the present invention include, for example, mannoproteins, β-glucans, ethanol, lactic acid, and other metabolites having bioemulsifying properties and surface / interfacial tension reducing properties.

[0133] At the time of recovering the bacterial-based composition from the growth vessel, when the recovered product is placed in a container and / or transported by pipe (or otherwise transported for use), additional components may be added. Additives may include, for example, dyes, pigments, pH adjusters, salts, adhesion promoters, chelating agents (e.g., EDTA, sodium citrate, citric acid), solvents (e.g., isopropyl alcohol, ethanol), biocides, other microorganisms, and other components specific to the intended use.

[0134] Advantageously, chelating agents enhance the effectiveness of the antimicrobial barrier composition by modifying, for example, the cell walls of Gram-negative bacteria, making them more susceptible to the effects of surfactant treatment. As a result, the spectrum of antimicrobial activity of the present invention is broadened by its ability to penetrate Gram-negative bacteria.

[0135] In one embodiment, the chelating agent is selected from EDTA, citric acid, citrate, sodium acetate, or any combination thereof. The chelating agent can be added to the composition in an amount of up to about 5 g / L or more. In a particular embodiment, the chelating agent is EDTA at a concentration of about 0.5 to 3 g / L.

[0136] Additives may be added as needed, for example, up to 50% by weight or more, for specific applications such as altering VOC levels, increasing the permeability of the mixture, lowering the viscosity of the mixture, acting as a coupler for insoluble substances in the mixture, and providing a solvent for lipophilic and hydrophilic soils.

[0137] In certain embodiments, the barrier composition of the present invention comprises a binder that primarily plays a role in binding pigments together in the coating. The binder may be an oil-based binder or a latex-based binder. The binder compound may be selected from, for example, acrylics, alkyds, acrylic acids, acrylamides, phenols, phenol-alkyds, polyacrylamides, polyurethanes, silicone-alkyds, polyesters, epoxys, vinyls, vinyl acetate-ethylene, vinyl-alkyds, inorganic binders (sodium, ethyl potassium silicate, lithium, etc.), organic binders (carbon-based), Tectyl® (Daubert Chemical Company, Inc., Chicago, IL), aliphatic urethanes, and oil-modified urethanes.

[0138] In certain embodiments, the barrier composition of the present invention comprises a pigment or dye that may provide color to a paint or other coating, and may further protect a surface or object from ultraviolet light. The pigment or dye may be natural, synthetic, inorganic, or organic. The pigment or dye may be selected from, for example, titanium dioxide, zinc oxide, zinc yellow, yellow dye, benzidine yellow, chromium oxide green, phthalocyanine green, phthalocyanine blue, ultramarine blue, vermilion, pigment brown 6, red 170, dioxazine violet, carbon black, iron(II) oxide, silica sand (SiO2), talc, barite (BaSO4), kaolin, and limestone (CaCO3).

[0139] In a particular embodiment, one of the solvents used in the composition is selected from inorganic or organic solvents, including, for example, ethanol, butanol, propanol, aliphatic hydrocarbons, alicyclic hydrocarbons, xylene, toluene, ketones, and / or isopropyl alcohol. In a preferred embodiment, isopropyl alcohol is added to the composition in an amount of 1 to 100 ml / L, more preferably 2 to 50 ml / L.

[0140] In certain embodiments, the composition further comprises an ionic or semiionic liquid as a solvent. The ionic liquid may act as a co-solvent and may prevent the formation of ring bonds in the hydrocarbon composition, which is one cause of hydrocarbon precipitation. Exemplary ionic liquids suitable for this composition include, but are not limited to, ethylammonium nitrate or glycerin / magnesium sulfate heptahydrate. Preferably, the concentration of the ionic liquid in the composition is in the range of about 0.1% to about 5%.

[0141] Ionic liquids are composed entirely of ions, which may include cations, anions, and / or combinations thereof. Many ionic liquids are in the form of organic salts with melting points below 100°C, or often even lower than room temperature. The most common ionic liquids are prepared from organic-based cations and inorganic or organic anions. At least one ion has a delocalized charge, and one component is organic, which prevents the formation of a stable crystal lattice. Ionic liquids are considered suitable for use as catalysts and solvents in reactions such as alkylation and polymerization, as well as in dimerization, oligomerization, acetylation, metathesis, and copolymerization. The properties of ionic liquids, such as melting point, viscosity, and solubility, are determined by substituents and counterions on the organic component.

[0142] In a particular embodiment, the composition further comprises ammonium hydroxide as a solvent. Preferably, ammonium hydroxide (70% solution) is present in the composition at a concentration of about 1 to 50 ml / L, more preferably 3 to 10 ml / L.

[0143] In one embodiment, a bacteria-based barrier product may further contain buffers comprising organic acids and amino acids or salts thereof. Suitable buffers include citrates, glucons, tartarates, malates, acetates, lactates, oxalates, aspartates, malons, glucoheptones, pyruvates, galactarates, glucarates, tartronates, glutamates, glycine, lysine, glutamine, methionine, cysteine, arginine, and mixtures thereof. Phosphoric acids and phosphites or salts thereof can also be used. Synthetic buffers are suitable for use, but natural buffers such as the organic acids and amino acids or salts thereof described above are preferred.

[0144] In a further embodiment, the pH adjuster includes potassium hydroxide, ammonium hydroxide, potassium carbonate or potassium bicarbonate, hydrochloric acid, nitric acid, sulfuric acid, or a mixture thereof.

[0145] Bacteria-based barrier products can be applied together with compositions that promote adhesion of the bacteria-based product to the surface to be treated. The adhesion promoter may be a component of the bacteria-based product, or it may be applied simultaneously with or consecutively with the bacteria-based product.

[0146] Other additives typically used in coating compositions may be used, including water softeners, metal ion sequestering agents, corrosion inhibitors, and antioxidants, which are added in amounts effective to perform their intended functions. These additives and their amounts are well within the scope of the art of the art. Suitable water softeners include linear phosphates, styrene-maleic acid copolymers, and polyacrylates. Suitable metal ion sequestering agents include 1,3-dimethyl-2-imidazolidinone; 1-phenyl-3-isoheptyl-1,3-propanedione; and 2-hydroxy-5-nonylacetophenone oxime. Examples of corrosion inhibitors include 2-aminomethylpropanol, diethylethanolamine benzotraizole, and methylbenzotriazole. Suitable antioxidants for the present invention include (BHT) 2,6-di-tert-butyl-para-cresol, (BHT) 2,6-di-tert-butyl-para-anisole, the Eastman inhibitor OAB M-oxalylbis(benzylidenehydrazide), and Eastman DTBMA 2,5-di-tert-butylhydroquinone.

[0147] In certain embodiments, the composition further comprises salts and / or inorganic salts selected from phosphorus, magnesium, potassium, glucose, and ammonium. Preferably, 1 to 20 g / L, and more preferably 2 to 10 g / L, of an ammonium salt, such as ammonium phosphate, diammonium phosphate, ammonium chloride, or another dibasic or monobasic salt is added.

[0148] Other suitable additives that may be contained in the formulations according to the present invention include substances conventionally used in such preparations. Additives may be, for example, carriers, other microbial-based compositions produced in the same or different facilities, viscosity modifiers, preservatives, tracking agents, biocides, desiccants, plasticizers, flow regulators, defoamers, emulsifiers, UV stabilizers, antidermatological agents, conditioners, emulsifiers, lubricants, solubility controllers, preservatives, and / or stabilizers.

[0149] Advantageously, according to the present invention, a bacteria-based product may include a broth in which microorganisms have grown. The product may be, for example, at least 1%, 5%, 10%, 25%, 50%, 75%, or 100% broth by weight. The amount of biomass in the product may range from 0% to 100% by weight, for example, including all proportions in between.

[0150] Optionally, the product can be stored before use. The storage time is preferably short. Therefore, the storage time may be less than 60 days, less than 45 days, less than 30 days, less than 20 days, less than 15 days, less than 10 days, less than 7 days, less than 5 days, less than 3 days, less than 2 days, less than 1 day, or less than 12 hours. In a preferred embodiment, if live cells are present in the product, the product is stored at a cold temperature, such as below 20°C, below 15°C, below 10°C, or below 5°C. On the other hand, bacterial-based compositions can typically be stored at ambient temperature.

[0151] The compositions according to the present invention may contain an effective amount of components for cleaning surfaces, structures, and equipment, and / or for providing an effective coating to prevent the future accumulation of contaminants, scale, and corrosion.

[0152] Use of microorganisms and their biofilm lysates in barriers The use of bacterial-derived barriers can bring about various improvements when applied to objects and / or surfaces.

[0153] Improved barrier performance and lifespan In certain embodiments, the application of the composition or method to an object improves the performance of a barrier, primarily through the modification of the interaction between the object and its surroundings. In certain embodiments, molecules (such as proteins) obtained from bacteria and / or lysates and / or bacterial biofilms can be applied to the surface of an object, which can prevent or limit direct contact by living or non-living organisms, prevent contamination by living or non-living organisms, isolate living or non-living organisms, inactivate living or non-living organisms, improve the durability of the barrier, and / or increase the concentration of symbiotic organisms. In some embodiments, the molecules are produced by synthesis and / or purified.

[0154] In a particular aspect, the present invention provides a self-contamination-free surface.

[0155] In certain embodiments, a composition can prevent direct contact with contaminants by physically separating the environment in which an object or surface resides from the object itself. Paints, varnishes, lacquers, glosses, viscosities, and other barriers provide physical separation between the environment within the barrier and the object.

[0156] In certain embodiments, the composition can prevent contamination by organisms such as viruses and bacterial cells. The composition can prevent surface adhesion and biofilm formation, thereby potentially limiting the transmission of disease-causing bacteria. The composition can also halt the growth of organisms or initiate apoptosis of cells.

[0157] In a particular embodiment, the composition of the present invention can prevent contamination by abiotic substances such as salt deposits.

[0158] In certain embodiments, the compositions of the present invention can improve the durability of barriers. This can be achieved by resisting frictional abrasion from liquids, gases, or solids.

[0159] In certain embodiments, the compositions of the present invention can increase the concentration of symbiotic organisms on an object or surface. This can be done indirectly by inhibiting the growth or adhesion of microbial competitors to the symbiotic organisms on the surface. Alternatively, the compositions can directly increase the growth rate of the symbiotic organisms.

[0160] The present invention can be used to prevent the occurrence of deposition. Dispersion or dissolution of biological or precipitated substances reduces the concentration of contaminants that may be present on a surface or object. Thus, the present invention makes it possible to delay or completely eliminate the need for preventive maintenance related to the removal of precipitates and deposits, as well as the need for replacement or repair of equipment parts. The coating composition can also be applied for the dispersion of scale buildup in storage and transport tanks, tankers, ships, trucks, pipelines and flow lines, concrete, asphalt, mulch, metal, siding, and stucco, for example, without the need for mechanical cleaning solutions or toxic solvents.

[0161] In a particular embodiment, the method is used to clean a surface, which is equipment requiring decontamination, defouling, and / or declogging. Advantageously, the method of the present invention can be used to improve the overall productivity of a production process or a single piece of equipment by improving equipment maintenance and proper functioning.

[0162] Application of the composition to the surface The composition can be applied to inanimate or living surfaces or objects such as skin, mucous membranes, clothing, metal surfaces, eyeglasses, protective clothing (including masks), and footwear.

[0163] In a particular aspect, the present invention provides a self-contamination-free surface.

[0164] In a particular aspect, the composition includes bandages; personal hygiene products; tableware and cooking utensils; tables and countertops; clothing including vests, shirts, trousers, socks, jackets, skirts, shorts, underwear, stockings, gloves, scarves, armor, diving suits, swimwear, spacesuits, formal wear, sportswear, leather clothing, and leisure wear; footwear including shoes, boots, sandals, slippers, fins, cleat shoes, deck shoes, wooden clogs, snow boots, ski boots, sailing boots, pointe shoes, and high heels; and brimped hats. It can be applied to headwear including children's helmets, crowns, brimless hats, bonnets, hoods, masks, turbans, veils, shawls, wigs, or medical devices; eyewear including eyeglasses, goggles, contact lenses, blindfolds, safety glasses, and sunglasses; and clothing accessories including bracelets, rings, bags, school bags, backpacks, handbags, necklaces, jewelry, watches, umbrellas, wallets, parasols, fans, swords, canes, ties, sashes, shawls, neck straps, decorative pins, earrings, and stockings.

[0165] In certain embodiments, the composition can be applied to vehicles including military vehicles, armored fighting vehicles, reconnaissance vehicles, self-propelled anti-aircraft guns, self-propelled air defense systems, ambulances, automobiles, trucks, jeeps, Humvees, vans, helicopters, bicycles, unicycles, scooters, skateboards, wagons, buses, hovercraft, and motorcycles; boats or warships including submarines, trawlers, drift net fishing vessels, patrol boats, destroyers, aircraft carriers, frigates, corvettes, battleships, battlecruisers, gunboats, and mine sweepers; trains including armored trains, passenger trains, freight trains, locomotives, maglev trains, monorails, and mining trains; aircraft including jet, propeller, or rocket propulsion types; and spacecraft including satellites, space probes, rockets, pods, capsules, and orbiters.

[0166] The composition can be applied to a surface, for example, by spraying it using a spray bottle or a pressurized atomizer. The composition can also be applied using a cloth or brush, in which case the composition is rubbed, spread, or brushed onto the surface. Furthermore, the composition can be applied to a surface by immersing, dipping, or submerging the surface in a container containing the composition.

[0167] In one embodiment, a substance and / or surface can be immersed in a composition for a time sufficient to apply a coating or to lift and / or remove contaminants from the object and / or surface. For example, the immersion may be carried out for 12, 24, 36, 48, 72 hours or longer, as needed.

[0168] In one embodiment, the method further includes a step of removing the composition and contaminants from the surface. This can be achieved, for example, by rinsing or spraying water on the surface and / or rubbing or wiping the surface with a cloth until the composition and contaminants are freed from the surface. Rinsing or spraying with water can be done before and / or after rubbing or wiping the surface with a cloth.

[0169] In another embodiment, contaminants and / or compositions can be removed from a surface by mechanical means. For example, a stirrer, drill, hammer, or scraper can be used to remove contaminants from a surface where removal is particularly difficult, for example, due to the amount or type of contaminants.

[0170] In certain embodiments, the composition may be administered in pharmaceutically acceptable forms, including by injection (i.e., subcutaneous, intravenous, intraperitoneal), oral, transdermal, or nasal.

[0171] material and method Bacterial strains and culture media Lactobacillus fermentum Qi6 (Lf Qi6) was grown in MRS medium at 37°C.

[0172] Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 (ATCC, Manassas, VA) was stored at -80°C in tryptone soy broth (TSB) (Thermo Scientific, Waltham, MA) containing 20% ​​(v / v) glycerol. The cultures were incubated aerobically overnight at 37°C on a 110 rpm rotary shaker. The optical density of the cultures overnight was read using a spectrophotometer (SpectraMax Plus384, Molecular Devices, Sunnyvale, CA), and the OD was 0.2. 600 The strain was diluted to the following degree. This strain was also subjected to both met and EAP PCR using established protocols to confirm the MRSA status (3).

[0173] Lf Qi6 Biofilm Culturing Lf Qi6 was cultured on an MRS agar plate. The culture was then incubated in 5 ml of MRS broth at 37°C for 24 hours. 1 ml of the culture was transferred to a T-150 tissue culture plate containing 25 ml of MRS broth. 25 ml of MRS medium was replaced every 48 hours to allow the Lf Qi6 biofilm to grow as a microbiota on the bottom of the culture plate. The culture was then grown for 7 days to form a thick biofilm layer. The grown biofilm layer was then detached and suspended in fresh medium. A frozen stock was prepared using glycerol and stored at -80°C.

[0174] Lf Qi6 in a biofilm phenotype from frozen storage was cultured in 10 ml of fresh MRS medium at 37°C for 24 hours. 10 ml of the culture was inoculated into 25 L of MRS medium containing 500 g of sterile glass wool. The biofilm was then cultured under static conditions at 37°C for 72 hours. The culture was mixed every 24 hours by gentle shaking, after which the medium and glass wool were collected. Subsequently, the biofilm cells were separated from the glass wool by sonication. The cells were further centrifuged to concentrate the Lf Qi6 biofilm, which was then suspended in sterile water. This scale-up yielded a biofilm culture with a concentration of 2 g / L.

[0175] Downstream processing of Lf Qi6 biofilm 50 g of Lf Qi6 biofilm was suspended in 1 L of sterile water. The suspension was gently mixed at room temperature for 24 hours to allow for the passive release of multiple bioactive substances. The mixture was then sonicated for 30 minutes (50 kHz, 200 watts) using an OmniSonic Ruptor 400 to obtain a homogeneous solution. The sonicated solution was then frozen and freeze-dried to obtain a fine powder.

[0176] Preparation of Lf Qi6 from probiotic bacteria L. fermentum Qi6 was grown in MRS medium using a proprietary culture method. The bacteria were then subcultured in 500 ml of MRS medium for an additional period, again using the same proprietary culture method. The bacteria were sonicated (Reliance Sonic 550, STERIS Corporation, Mentor, OH, USA), centrifuged at 10,000 × g, the cell pellet was dispersed in sterile water, the recovered cells were lysed (Sonic Ruptor 400, OMNI International, Kennesaw, GA, USA), and the soluble fraction was centrifuged again at 10,000 × g (50 kDa Amicon ultrafiltration membrane, EMD Millipore Corporation, Darmstadt, Germany, Cat#UFC905008). The resulting fraction was dispensed into 0.5 ml portions, flash-frozen in liquid nitrogen, and stored at -80°C.

[0177] Biofilm Inhibition Assay MRSA was added to the wells of sterile polystyrene, tissue culture (TC) treated, flat-bottom plates (Genesee Scientific, San Diego, CA, Cat #25-109). TSBs were used as sterile controls. Equal volumes of MRSA and culture medium were added to the growth control wells. Selected concentrations of Qi601S or other test reagents were added, and the plates were incubated at 37°C for 18 hours, after which the biofilms were quantified as described in the staining and biofilm quantification sections.

[0178] Staining and biofilm quantification Tissue culture plates were washed three times with PBS using a BioTek plate washer, and the biofilms were heat-fixed in a 47°C incubator for 1 hour. The plates were cooled to room temperature, stained with 0.1% (v / v) crystal violet for 15 minutes, and then washed with deionized H2O using a microplate washer. 100% ethanol was added to dissolve the crystal violet stain and allowed to stand for 30 minutes. The plates were read at 590 nm and 600 nm using a spectrophotometer (SpectraMax Plus384, Molecular Devices, Sunnyvale, CA). [Examples]

[0179] A deeper understanding of the present invention and its many advantages can be obtained from the following examples provided for illustrative purposes. The following examples illustrate several methods, applications, aspects, and variations of the present invention. They should not be considered limiting to the present invention. Numerous changes and modifications can be made with respect to the present invention.

[0180] Example 1 - Qi601SL, Qi601SML, Qi601A, Qi601AM, and Qi601SP inhibit MRSA adhesion (Figures 2, 6, 7, and 8). To demonstrate that various versions of lysates from Lactobacillus fermentum Qi6 can inhibit MRSA adhesion, MRSA was first grown to the logarithmic phase, diluted with TSB, and then various forms of Qi601S (Qi601SL, Qi601SML, Qi601SP, Qi601AM, or Qi601A) were added to the MRSA in 96-well or 24-well plates. The cultures were incubated overnight. Each sample was visualized for adhesion using a plate reader that stained crystal violet and measured light absorption at 590 nm.

[0181] Lf Qi601 lysates can inhibit MRSA adhesion, thereby establishing a novel method for inhibiting MRSA biofilm adhesion.

[0182] Example 2 – Qi601SL, Qi601SP, Qi601SML, Qi601A, Qi601AM, and Qi601SMP continue to inhibit MRSA adhesion even after washing (Figures 1, 3, 4, 5, 7, and 8). To demonstrate that various versions of lysates from Lactobacillus fermentum Qi6 can inhibit MRSA adhesion, MRSA was first grown to the logarithmic phase, diluted with TSB, and then various forms of Qi601S (Qi601SL, Qi601SML, Qi601SMP, Qi601SP, Qi601A, or Qi601AM) were added to the MRSA in 96-well or 24-well plates. The cultures were incubated overnight. Each sample was visualized for adhesion using a plate reader that stained crystal violet and measured light absorption at 590 nm.

[0183] As demonstrated in both Example 1 and Example 2, the ability of the dissolve to inhibit MRSA adhesion is significant, and the durability of the composition is demonstrated by the dissolve's ability to maintain its effect even after washing.

[0184] Example 3 - Atomic Force Microscopy Figure 9 provides an atomic force microscope image of the material of the present invention.

[0185] Example 4 - Prevention of pathogen attachment Pathogen adhesion to human tissue is essential for the pathogenicity of Escherichia coli O157:H7, a foodborne pathogen. Therefore, we evaluated the anti-adhesion effect of Qi 601S on human intestinal cells against Escherichia coli, a model gram-negative pathogen. These results in human cells (Figure 10) demonstrate that treatment with Qi601S creates an E. coli-resistant anti-adhesion surface.

[0186] Figure 10 shows how Qi 601S creates an effective anti-adhesion surface against E. coli on Caco-2 cells. (5 × 10 cells per well) 4The cells were divided into 24-well plates and grown for a further 10 days for differentiation and concentration. Selected wells were pretreated overnight with Qi 601S 0.1% v / v and then washed with PBS. E. coli K12 was grown overnight in TSB, and 1 × 10⁶ cells were grown. 8 CFU was added to each well and incubated for 3 hours. Unbound bacteria were removed by PBS washing. Intestinal cells were lysed with 1% Triton X-100. Binding bacteria were quantified by serial dilution and standard plate count in TSA and compared to the untreated control.

[0187] The durability of Qi 601S's antimicrobial and anti-adhesive (self-cleaning) activity over 48 hours was also demonstrated in living human tissue. To demonstrate in vivo protection, human ex vivo full-thickness skin specimens (excess tissue from selective abdominoplasty obtained immediately after removal) were used.

[0188] In short, the method was as follows: Ex vivo skin was pre-confirmed to be MRSA-negative by standard culture methods. Colonies were phenotypically identified on MRSA-selective medium (Chromagar, Becton Dickinson) according to the package instructions. Abdominal wall reconstruction skin was maintained in serial culture and used within two weeks of surgical excision after a 3-day medium change to remove residual preoperative antibiotics. One centimeter punch biopsy specimens were obtained from single donor exgraft tissue and five replicas were experimented with in 24-well plates.

[0189] The epidermis was treated with a medium (300 µl PBS) or Qi601S (1% in 300 µl PBS) for 24 hours. The skin was then rinsed three times with PBS and placed in a 0.5 micron transwell insert in a new culture plate. The experimental sample was then cultured in an OD=0.1 (1 × 10⁻¹⁶) state. 9The samples were incubated with 5 µl MRSA (CFU / ml) for 48 hours, rinsed again with PBS to remove non-adherent bacteria, and transferred to a Petri dish. Each skin sample was swabded three times with a cotton swab, and the tip of the swab, along with a glass bead, was placed in a tube containing 0.1% Triton-X in PBS, then vortexed for 15 seconds. Serial dilutions were plated on Luria Bertani (LB) agar and Chromagar, and colonies were counted the following day.

[0190] Figure 11 shows the anti-adhesion surface against MRSA generated by Qi 601S. 1% v / v Qi 601S in PBS was applied to live human skin in culture for 24 hours, followed by rinsing. The skin was then cultured with MRSA for 2 days, the skin was rinsed, and then MRSA colonies were cultured. Pretreatment with Qi 601S demonstrated a significant reduction in MRSA load of over 60% compared to untreated skin.

[0191] Example 5 - Defense against biological threats In addition to its activity against MRSA biofilms, Qi 601S also demonstrated activity against other biological threats, including viral threats. Biological weapons are classified by the CDC into three categories based on the risks of bioterrorism defense, biological warfare, and bioterrorism. Category A includes readily transmissible or contagious biofactors such as anthrax; Category B includes moderately transmissible or contagious biofactors such as Salmonella, Escherichia coli O157:H7, and Staphylococcus aureus, particularly MRSA; and Category C includes emerging factors. Category C is continuously reassessed. For example, coronaviruses were added to Category C in 2014 by the NIAID, CDC, and the U.S. Department of Homeland Security, and Category C now includes other highly pathogenic human coronaviruses, including Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV), MERS-CoV, and COVID-19 (NIAID Emerging Infectious Diseases / Pathogens, 2018).

[0192] Qi 601S also demonstrated complete protection against acetylcholinesterase (AChE) inhibition. This enzyme is crucial for normal neuronal responses and function, and its inhibition is a target for nerve gases, which are irreversible AChE inhibitors. Irreversible inhibition of this enzyme leads to hyperexcitation of the nervous system, ultimately resulting in death. AChE breaks down the excitatory neurotransmitter acetylcholine (Ach) into choline and acetate at the synapses of nerve cells and is primarily found in cholinergic synapses of the central nervous system.

[0193] Organophosphates (OPs) are used as insecticides and nerve agents because they are lethal in microgram amounts. These chemicals are among the most toxic compounds known, yet there are currently no non-destructive methods to decontaminate exposed victims.

[0194] The potential of Qi601S for AChE activity protection was tested against donepezil enzyme inhibitory activity using a standard assay kit (Abcam, #138871). Figure 13 shows the results, demonstrating potent, complete, and consistent levels of AChE protection by 2% Qi601S in response to escalating doses of donepezil hydrochloride.

[0195] Example 6 - Activity and Stability The safety profile of the composition of the present invention enables biomedical applications as a self-decontamination agent, applied topically to the skin, nose, and eyes, and inhaled into the lungs. A single application of a 1% solution of the heat-stable hydrophilic fraction Qi 601S provides non-toxic anti-coronavirus protection in an in vitro SARS-CoV model for more than 5 days, and durable anti-MRSA anti-adhesion on inert hydrophobic surfaces (polypropylene and glass) for up to 2 days (at the last time tested), and despite repeated rinsing, over a pH range of at least 4.5–7.5, even with a small amount of pH. Unlike many biological products, this fraction maintains stability when exposed to temperatures above 121°C, repeated freeze-thaw cycles, and aqueous media. The maintenance of anti-MRSA efficacy when tested as an ammonium sulfate precipitate, and compositional analysis of the chemically purified biosurfactant, indicate that the antimicrobial and surfactant activities are protein-derived.

Claims

1. A composition comprising a Lactobacillus species grown as a biofilm and / or an extract of said biofilm, wherein, when the composition is applied to a surface, the biofilm and / or extract enhance the barrier function such that the surface becomes less susceptible to the effects of microorganisms, viruses, and / or chemical contamination.

2. The composition according to claim 1, wherein the bacterial strain is L. fermentum Qi6 having accession number PTA-122195.

3. The composition according to claim 1, comprising a heat-stable protein produced by the Lactobacillus genus.

4. below: i) SEQ ID NO: 1; ii) Variants of SEQ ID NO: 1; and iii) Fragment of i) or ii) The composition according to claim 1, comprising a protein containing a more selected amino acid sequence.

5. A surface to which the composition according to claim 1 is applied.

6. The surface according to claim 5, which is the skin, intestines, stomach, lungs, eyes, mouth, sinuses, nose, ears, trachea, or esophagus.

7. The surface according to claim 5, which is on an object selected from medical devices, countertops, protective clothing, tables, electrical appliances, chairs, tableware, cooking utensils, medical implants, bandages, personal hygiene products, clothing, eyeglasses, footwear, headwear, and masks.

8. The surface according to claim 5, which is located on an object selected from vehicles, boats, ships, automobiles, trucks, tanks, aircraft, and trains.

9. The method according to claim 5, wherein the composition applied to the surface comprises L. fermentum Qi6 having accession number PTA-122195.

10. The method according to claim 5, wherein the composition applied to the surface comprises a heat-stable protein produced by the Lactobacillus genus.

11. The compositions applied to the surface are as follows: i) SEQ ID NO: 1; ii) Variants of SEQ ID NO: 1; and iii) Fragment of i) or ii) The method according to claim 5, comprising a protein having a more selected amino acid sequence.

12. A method for enhancing a surface's barrier against chemical, viral, and / or microbial contamination, comprising the step of applying the composition according to claim 1 to the surface.

13. The method according to claim 12, wherein the surface is selected from the skin, intestines, stomach, lungs, eyes, mouth, nose, ears, trachea, or esophagus of the subject.

14. The method according to claim 12, wherein the surface is selected from medical devices, countertops, protective clothing, tables, electrical appliances, chairs, tableware, cooking utensils, medical implants, personal hygiene products, bandages, clothing, eyeglasses, footwear, headwear, and masks.

15. The surface according to claim 12, wherein the inanimate surface is located on an object selected from vehicles, boats, ships, automobiles, trucks, tanks, aircraft, and trains.

16. The performance and / or lifespan of an object and / or surface are as follows: a) Preventing or limiting direct contact between a surface and living or non-living entities; b) To prevent contamination of surfaces by living or non-living things; c) Isolating living or non-living things; d) Inactivating living or non-living things; e) To enhance the durability, stability, and / or lifespan of the barrier; and e) Increasing the presence of symbiotic organisms The method according to claim 12, further improved by one or a combination of the following.

17. The method according to claim 12, wherein the composition applied to the surface comprises L. fermentum Qi6 having accession number PTA-122195.

18. The method according to claim 12, wherein the composition applied to the surface comprises a heat-stable protein produced by the Lactobacillus genus.

19. The compositions applied to the surface are as follows: i) SEQ ID NO: 1; ii) Variants of SEQ ID NO: 1; and iii) Fragment of i) or ii) The method according to claim 12, comprising a protein having a more selected amino acid sequence.

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