Composition and method for modulating microbiomes with postbiotic ferments

Abstract

A microbiome modulating composition and a method for producing the microbiome modulating composition are described herein. The method comprises preparing a culture from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof, passaging the culture on a glycogen carbon source to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof, and adding the postbiotic ferment to a carrier. Further disclosed is a method for treating microbial dysbiosis of an urogenital area or a skin area of a user, the method comprising applying the microbiome modulating composition to an urogenital or a skin area of a user. The disclosed methods and microbiome modulating compositions help promote the growth of healthy Lactobacillus spp. bacteria to prevent dysbiosis and other related conditions and infections.

Description

[0001] COMPOSITION AND METHOD FOR MODULATING MICROBIOMES WITH POSTBIOTIC FERMENTS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to the benefit of U.S. Provisional Application No. 63 / 717,995, filed November 8, 2024, which is expressly incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] Humans are colonized by microbes in the gastrointestinal tract, on the skin, and in other epithelial and tissue niches such as the oral cavity, eye surface and vagina. Healthy microbial flora in these areas can comprise hundreds of different species of bacteria. A healthy microbial flora provides the host with multiple benefits, including colonization resistance to a broad spectrum of pathogens, essential nutrient biosynthesis and absorption, and immune stimulation. A marker of a healthy microbial flora is often the presence of known healthy Lactobacillus spp. bacteria. Lactobacillus spp. bacteria are commonly found on outer surfaces of the body, such as skin, epithelium, and mucous membranes. The presence of Lactobacillus spp. bacteria has been established to be important in the regulation of urogenital health, especially for women, as Lactobacillus spp. bacteria are common in the vagina and bladder.

[0006] For example, a normal vagina generally contains more than about 104lactobacilli per milliliter of vaginal fluid. Under normal conditions, the vagina flora provides a mildly acidic environment that helps guard against the invasion of pathogenic microbes. Unfortunately, the balance of the vagina flora may be easily upset by a variety of external factors that ultimately lead to vaginal infection. Vaginal infections include, but are not limited to, bacterial vaginosis, candidal vaginitis ("yeast"), and trichomonas vaginitis ("trich") . Bacterial vaginosis, for instance, is one of the most common dysbiotic conditions of the vaginal environment that is characterized by an increase in pH, discharge, odor, and discomfort. Bacterial vaginosis is often driven by a significant reduction of healthy vaginal flora combined with an outgrowth of pathogenic bacteria including bacteria from the Streptomyces, Streptococcus, Gardnerella, Escherichia, Prevotella, Candida, Staphylococcus, and Enterococccus genera.

[0007] Current treatment regimens for a bacterial infection of the vagina involve the use of various broad-spectrum antibiotics, such as metronidazole. However, antibiotics are often undesirable because they may kill a broad range of the normal bacterial flora in the vagina, including the beneficial lactobacilli. This may cause secondary complications, because the lactobacilli help regulate various other opportunistic pathogens in the vagina. The treatment may then necessitate a further treatment regimen, such as the ingestion of cultured dairy products to replace the lactobacilli in the body, as well as treatment by antifungal agents. Moreover, a rise in the level of anaerobes due to a lack of lactobacilli could further complicate the infection. Additionally, antibiotics, when used frequently within the vagina, may cause systemic toxicity through absorption from the vagina.

[0008] Accordingly, a need exists for compositions and methods for modulating microbiomes, especially the microbiomes of the vagina, bladder, and other skin areas to improve urogenital health in a user. A need also exists for compositions and methods for preventing bacterial vaginosis, candidal vaginitis, and trichomonas vaginitis in a user.

[0009] SUMMARY OF THE DISCLOSURE

[0010] In general, the present disclosure is directed towards a method and composition for modulating microbiomes, particularly urogenital and skin microbiomes associated with women’s health, with postbiotic ferments of Lactobacillus spp. and Limosilactobacillus spp. bacteria. For instance, the method comprises preparing a culture from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof and passaging the culture on a glycogen carbon source to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof. The method further comprises adding the postbiotic ferment to a carrier. In some example embodiments, the microbiome modulating composition is buffered to have a pH of from about 3.8 to about 5.0 to be biologically compatible with urogenital areas, such as the vaginal canal.

[0011] In other example embodiments, the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof include, but are not limited to, a mixture of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillus johnsonii, Limosilactobacillus vaginalis, Limosilactobacillus reuteri, Limosilactobacillus fermentum, or Limosilactobacillus rhamnosus. In certain example embodiments, the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof include a mixture of Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus iners.

[0012] In some example embodiments, a probiotic comprising a mixture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof is further added to the microbiome modulating composition. In other example embodiments, a prebiotic is added to the microbiome modulating composition. In preferred embodiments, the prebiotic comprises at least one Lactobacillus spp. bacteria-specific prebiotic, at least one Limosilactobacillus spp. bacteria-specific prebiotic, or combinations thereof.

[0013] In certain example embodiments, one or more additional steps can be employed to manipulate the production of the postbiotic ferment. For instance, in some example embodiments, the culture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof are initially prepared on De Man-Rogosa-Sharpe (MRS) culture media with glucose. In other example embodiments, the glycogen carbon source is a Minimal Lactobacillus Media (MLM) culture media without glucose. In certain example embodiments, the method further comprises pelleting the culture to form a pellet, washing the pellet with 2 x PBS solution, and re-passaging the culture on a glycogen carbon source. In other example embodiments, the culture was passaged on the glycogen carbon source under hypogravity conditions.

[0014] The present disclosure is also generally directed towards a microbiome modulating composition comprising a carrier and a postbiotic ferment produced from passaging a culture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof on a glycogen carbon source. In some example embodiments, the carrier is an aqueous solution. Thus, in other example embodiments, the microbiome modulating composition is a gel, a liquid, a cream, a spray, or a suppository. In preferred example embodiments, the microbiome modulating composition is a vaginal insertable gel.

[0015] In certain example embodiments, the postbiotic ferment comprises about 0.1 % wt. / vol to about 10% wt / vol of the microbiome modulating composition. In other example embodiments, the carrier comprises greater than about 90% wt / vol of the microbiome modulating composition. Yet, in other example embodiments, the microbiome modulating composition may include one or more additional components. For instance, in certain example embodiments, the microbiome modulating composition further comprises a probiotic comprising a mixture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof. In other example embodiments, the microbiome modulating composition further comprises a prebiotic, wherein the prebiotic comprises at least one Lactobacillus spp. bacteria-specific prebiotic, at least one Limosilactobacillus spp. bacteria- specific prebiotic, or combinations thereof. In certain example embodiments, the microbiome modulating composition further comprises at least one of an additional urogenital prebiotic, a surfactant, an ester, a humectant, a pH adjuster, a rheology modifier, a gelling agent, a nitrogen source, and an antimicrobial agent

[0016] The present disclosure is also generally directed towards a method for treating microbial dysbiosis of an urogenital area or a skin area of a user. The method comprises applying a microbiome modulating composition to an urogenital or a skin area of a user, the composition comprising a carrier and a postbiotic ferment produced from passaging a culture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof on a glycogen carbon source. In some example embodiments, the method further comprises applying the microbiome modulating composition to a substrate, the substrate comprising a wipe or at least a portion of an absorbent article. In other example embodiments, the method further comprises promoting a healthy microflora balance of the urogenital area or the skin area of the user, wherein promoting the healthy microflora balance of the urogenital area or the skin area of the user comprises promoting a growth of Lactobacillus spp. bacteria relative to a growth of one or more types of Enterococcus spp. bacteria, Staphylococcus spp. bacteria, Escherichia spp. bacteria, Gardnerella spp. bacteria, Streptococcus spp. bacteria, or mixtures thereof. For instance, in some example embodiments, when the microbiome modulating composition is applied to a culture of Lcrispatus on Minimal Lactobacillus Media (MLM) culture media, as described below, the culture of L. crispatus demonstrates a lag time in growth of about 105 minutes or less. In other example embodiments, when the microbiome modulating composition is applied to a culture of Lcrispatus on Minimal Lactobacillus Media (MLM) culture media, the culture of L crispatus demonstrates a growth rate during exponential growth of about 0.004 AOD / min or greater. In other example embodiments, when the microbiome modulating composition is applied to a culture of Lcrispatus on Minimal Lactobacillus Media (MLM) culture media, a final bacterial concentration (OD at 600 nm) of L crispatus is about 1 .55 or greater. Yet in other example embodiments, when the microbiome modulating composition is applied to a culture of E. faecalis on Minimal Lactobacillus Media (MLM) culture media, the culture of E. faecalis demonstrates a growth rate during exponential growth of about 0.0006 AOD / min or less. In certain other example embodiments, when the microbiome modulating composition is applied to a culture of E. faecalis on Minimal Lactobacillus Media (MLM) culture media, the final bacterial concentration (OD at 600 nm) of E.faecalis is about 0.3 or less.

[0017] These and other features, aspects and advantages of the present disclosure will become better understood with reference to the following description and appended claims. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and, together with the description, serve to explain the principles of the disclosure.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] A full and enabling disclosure of the present disclosure is set forth more particularly in the remainder of the specification, including reference to the accompanying figures, in which:

[0020] Figure 1 graphically illustrates the production of the microbiome modulating gels including either a postbiotic ferment (e.g. “postbiotic”) or a postbiotic ferment and probiotic (e.g. , “postbiotic / probiotic”) as described in Example 1.

[0021] Figure 2 graphically illustrates the growth of L. crispatus in the presence or absence of postbiotic extract or postbiotic / probiotic extract described in Example 2. Figure 3 graphically illustrates the growth of E. faecalis in the presence or absence of postbiotic extract or postbiotic / probiotic extract as described in Example 2.

[0022] Figure 4 graphically illustrates the optical density as a function of time for the postbiotic / probiotic extract gel in comparison to a control gel that only contained culture media as described in Example 2.

[0023] Figure 5 graphically illustrates the growth of L. crispatus in the presence or absence of probiotic additives to the postbiotic extract as described in Example 3.

[0024] Figure 6 graphically illustrates the growth of L crispatus in the presence of the postbiotic / probiotic extract that was prepared with or without passage on glycogen as the sole carbon source as described in Example 4.

[0025] Figure 7 graphically illustrates the growth of E. faecalis in the presence of the postbiotic / probiotic extract that was prepared with or without passage on glycogen as the sole carbon source as described in Example 4.

[0026] Repeat use of reference characters in the present specification and the drawings is intended to represent same or analogous features or elements of the invention.

[0027] DEFINITIONS

[0028] As used herein, the term “inhibit” generally means to reduce by a measurable amount or to prevent entirely.

[0029] As used herein, the term “urogenital” refers to the vaginal canal, vulva, vagina, urethra, urinary tract, bladder, perineal, anal, and surrounding areas.

[0030] As used herein, the term “skin” refers to epithelium, mucous membranes, and any other tissue forming the outer layer of a body’s surface.

[0031] As used herein, the term “user” or “subject” refers to a person who is receiving the composition of the present disclosure.

[0032] As used herein, the terms “effective amount” and “therapeutic amount” is an amount sufficient to inhibit, but not necessarily kill, pathogenic microorganisms that may be responsible for or lead to infection of the vagina or bladder. In fact, although not required, it may be desired to use a concentration that does not significantly affect or inhibit the growth characteristics of the normal vagina or bladder flora or otherwise significantly irritate the vaginal or bladder tissue when used at inhibitory, noncytotoxic, or clinical concentrations. For example, a therapeutic agent can be desirably employed at a concentration of about 0.01 % to about 10% wt / vol, in some embodiments from about 0.1 % to about 7.5% wt / vol, in some embodiments from about 0.2% to about 5.0% wt / vol, and in some embodiments from about 0.5% to about 2.5% wt / vol. It should be understood that the dosage may vary with the age, condition, and microbiome of the site, and may be readily determined by one of skill in the art.

[0033] As used herein, the term “therapeutic effect” refers to the ability of the compositions and methods of the present disclosure to stimulate the growth of Lactobacillus spp. bacteria relative to deleterious bacteria according to the therapeutic effect protocol described below. The therapeutic effect can be expressed as a ratio of Lactobacillus spp. bacteria growth to Enterococcus faecalis growth when exposed to a postbiotic ferment, either alone or in further combination with a probiotic, is about 4 to about 12, such as about 5 to about 11 , such as about 5.5 to about 10.5.

[0034] As used herein, the designation “% wt / vol” or “wt / vol” refers to the value obtained by dividing the weight of a substance (in grams) by the volume of the solution (in milliliters) and then multiplying by 100.

[0035] As used herein the term "soluble" when having reference to a therapeutic agent means that the substance is at least soluble according to the method described by L. Prosky et al, J. Assoc. Off. Anal. Chem. 71 , 1017-1023 (1988)

[0036] As used herein the term "ferment” refers to the metabolic by-products and components produced by microorganisms, such as bacteria, during fermentation. Thus, ferments may include byproducts of fermentation, components of bacteria, including, but not limited to, cell wall fragments, membrane components, surface proteins and peptides, extracellular polysaccharides, DNA fragments, RNA fragments, and any other fermentation products.

[0037] DETAILED DESCRIPTION OF THE DISLOSURE

[0038] A composition and method for modulating microbiomes with a postbiotic ferment (e.g., “postbiotics”) is provided herein. Specifically, the composition and method for producing the postbiotic ferments as disclosed herein are particularly suitable for modulating the microbiomes of an urogenital area or a skin area of a user. The postbiotic ferments of the present invention are formed from passaging a culture of a mixture of Lactobacillus spp. bacteria or Limosilactobacilius spp. bacteria on a glycogen carbon source. Without intending to be limited by theory, the present inventors have found that a mixture of at least two of any Lactobacillus spp. bacteria or Limosilactobacilius spp. bacteria may function as a “mini-microbiome,” and thus produce more effective postbiotic ferments that can stimulate the growth of Gram-positive rod-shaped bacteria belonging to the Lactobacillus spp. It is believed that stimulating the growth of, and dominance of, lactobacilli may reestablish healthy flora by reducing or excluding the population of deleterious, pathogenic bacteria. Notably, the present inventors have discovered that utilizing a composition including a postbiotic ferment formed from a mixture of Lactobacillus spp. bacteria or Limosilactobacillus spp. bacteria passaged on a glycogen carbon source can be utilized to preventatively treat infections, such as reoccurring infections, post-intercourse infections, or alternatively could be used to treat existing infections such as urinary tract infections, bacterial vaginosis, etc. Further, the composition and the methods utilizing the composition disclosed herein may be significantly safer than repeated antibiotic use, as repeated antibiotic use often deplenishes normal bacterial flora, including the beneficial lactobacilli.

[0039] Lactobacillus spp. help to promote a healthy intimate microenvironment through competitive exclusion of pathogens, production of hydrogen peroxide, production of bacteriocins, production of surfactants, and production of other antimicrobial products that prevent infection and train the immune system. Notably, lactobacilli produce lactic acid, which can help lower the vaginal pH. Further, lactic acid can also play a role in maintaining the skin barrier. Accordingly, the methods and compositions disclosed herein have broad applicability, and in some embodiments, could be applied anywhere lactobacilli are present. For instance, although the compositions of the present disclosure may be applied to an urogenital area of a user to help maintain a healthy intimate microenvironment, the composition of the present disclosure may also be applied broadly to the skin and can help promote overall skin health, reduce skin inflammation, and treat common skin conditions caused by microbiome dysbiosis, such as acne.

[0040] The method for producing a microbiome modulating composition as described herein includes preparing a culture from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof, passaging the culture on a glycogen carbon source to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof, and adding the postbiotic ferment to a carrier. The carrier of the composition can include, for instance, a “dermatologically acceptable carrier”, which refers to a carrier that is suitable for topical application to epithelium and is compatible with the postbiotic ferment. The dermatologically acceptable carrier may be in a wide variety of forms such as, for example, simple solutions (water-based or oil-based) and solid forms (e g., gels or sticks). In some embodiments, the carrier can be either aqueous or nonaqueous. Non-aqueous carriers may include, for example, glycols, such as propylene glycol, butylene glycol, triethylene glycol, hexylene glycol, polyethylene glycols, ethoxydiglycol, and dipropyleneglycol; alcohols, such as ethanol, n-propanol, and isopropanol; triglycerides; ethyl acetate; acetone; triacetin; and combinations thereof. However, in some embodiments, the non-aqueous carrier is not an alcohol such that the composition is free from an alcohol.

[0041] In preferred embodiments, the carrier is an aqueous solution and the microbiome modulating composition may be in the form of a vaginally inserted gel. For instance, water is a particularly preferred aqueous carrier, such that the dermatologically acceptable carrier is used as a water-based simple solution. In some embodiments, the carrier constitutes greater than about 75% wt / vol, more preferably greater than about 85% wt / vol, and still more preferably greater than about 90% wt / vol. In other embodiments, the carrier can constitute greater than about 95% wt / vol, or greater than about 96% wt / vol, 97% wt / vol, 98% wt / vol, or even 99% wt / vol.

[0042] Lactobacillus spp. are used throughout the present disclosure for the production of the postbiotic ferments and as probiotic compounds that are optionally added to the microbiome modulating composition. For instance, the present inventors have found that the postbiotic ferments have a therapeutic effect on promoting the growth of Lactobacillus spp. bacteria and diminishing the growth of deleterious bacteria even when used as the sole therapeutic agent in the microbiome modulating composition. Without intending to be limited by theory, however, the present inventors have found that utilizing a mixture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof help produce more effective postbiotic ferments than if only a single Lactobacillus spp. or Limosilactobacillus spp. bacteria were utilized to form the postbiotic ferments. In certain embodiments, the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof used to form the postbiotic ferments include, but are not limited to, a mixture of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillus johnsonii, Limosilactobacillus vaginalis, Limosilactobacillus reuteri, Limosilactobacillus fermentum, or Limosilactobacillus rhamnosus. Potential concentration ranges are between about 1 x 104to about 1 x 1010CFU / mL, such as about 1 x 106to about 1 x 108CFU / mL. Of course, it should be understood by one of ordinary skill in the art that any bacteria from the Lactobacillus spp. or Limosilactobacillus spp. may be suitable for the microbiome modulating composition. In preferred embodiments, the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof include a mixture of Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus Iners. In other preferred embodiments, about 106CFU / mL of each Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus iners were utilized to prepare the postbiotic ferments.

[0043] However, it should be understood that because microbiome dysbiosis differs from person to person, the method for forming the postbiotic ferment and resulting microbiome modulating composition of the present disclosure may also have particularly suitable applications in preparing personalized postbiotic ferments. For instance, personalized postbiotic ferments may be created using a minimicrobiome harvested from a single individual for eventual treatment in that same individual. As Lactobacillus spp. bacteria are frequently found in urogenital areas, such as the vaginal canal, or on other parts of the skin, the starting composition of Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof may be selected based on an individual’s ''mini-microbiome,'' such that the resulting microbiome modulating composition is best tailored to support or treat their personal flora.

[0044] In certain example embodiments, cultures from the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof are initially prepared on De Man-Rogosa- Sharpe (MRS) culture media with glucose. As described in further detail below, the culture of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof are then centrifuged to form a pellet. The pellet is washed with 2 x PBS solution and passaged or sub-cultured on a glycogen carbon source.

[0045] Glycogen carbon sources include any form of culture media that are glycogen rich, and thus, may replicate the glycogen-rich microenvironment experienced by the bacteria in the vaginal cavity. Glycogen carbon sources may include both naturally derived and bio-tech derived glycogen. For instance, sources of glycogen include, but are not limited to, glycogen derived from oysters and other shellfish or from mammalian muscle or liver. Other suitable sources of glycogen include other animal sources, such as mammalian kidneys, heart, red and white blood cells, uterus, and glial cells in the brain. However, it should be understood by one of ordinary skill in the art that glycogen can be added to any favored growth media to form a glycogen carbon source. Thus, in some embodiments, the glycogen carbon source may include De Man, Rogosa, Sharpe (MRS) broth, Vaginal Fluid Simulating Medium (VFS), Minimal Lactobacillus Media (MLM) broth, and any other glycogen-supplemented media as known in the art.

[0046] In preferred embodiments, the glycogen carbon source is a Minimal Lactobacillus Media (MLM) culture media without glucose. In some embodiments, the Minimal Lactobacillus Media (MLM) culture media is composed of 25 mM MOPS (pH 6.8), M9 minimal salts ( 1 X), Sigma V1 Vitamin Kit, and Amino Acids Mix Solution (1X). It is further supplemented with 1 mM MgCl2-6H2O, 1 mM MnCl2-4H2O, 1mM FeSO4- 7H2O, 250 piM tryptophan, 250 piM cysteine, 250 piM CaCL, 250 piM ZnSO4-7H2O, 50 piM CUSO4-5H20, 1 mM TWEEN 80-, and 125-mM bovine glycogen (or 125 mM glucose). Guanine, thymine, cytidine, 2’-deoxyadenosine, and 2’-deoxyuridine were added to a final concentration of 0.5 mM each.

[0047] In other example embodiments, the method for producing the microbiome composition may include one or more additional steps. For instance, the method may further comprise pelleting the culture of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof in the Minimal Lactobacillus Media (MLM) broth to form a pellet, washing the pellet with 2 x PBS solution, and re-passaging the culture on a glycogen carbon source. Thus, in some example embodiments, the culture of the at least two Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof may be subjected to about four or more passages. In other example embodiments, the Lactobacillus spp. bacteria, the Limosilactobacillus spp. bacteria, or combinations thereof may be passaged or subcultured on MRS culture media (+) glucose, MRS culture media (-) glucose (+) glycogen, and Minimal Lactobacillus Media (MLM) culture media (-) glucose (+) glycogen.

[0048] However, it should be understood by one of ordinary skill in the art that other suitable culture media or subculturing techniques may be utilized to manipulate certain characteristics and effects of the resulting postbiotic ferments. For instance, in one example embodiment, the culture may be passaged on the glycogen carbon source in a reduced gravity environment. Notably, the reduced gravity environment may maintain a gravitational load of an average gravity value of about 0.0 x g to about 0.25 x g, such as about 0.0 x g to about 0.005 x g, such as about 0.0012 x g to about 0.0042 x g. Without intending to be limited by theory, the present inventors believe that passaging the bacteria under hypogravity conditions may affect the metabolites of the resulting postbiotic ferments prepared under hypogravity conditions (i.e. “hypogravity postbiotic ferments”) which may result in improved efficacy in both promoting the growth of healthy, beneficial bacteria, and inhibiting the growth of deleterious or pathogenic spp. compared to postbiotic ferments prepared under "normal” gravitational loads of about 1 .0 x g.

[0049] The method further comprises harvesting the supernatant of the pelleted culture passaged on a glycogen carbon source and further subjecting it to high-speed centrifugation and Sephadex G-10 column (10 cm x 3 cc2) fractionation to obtain a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof. As referred to herein, postbiotic ferments may include soluble and insoluble products secreted by or made of Lactobacillus spp. bacteria or Limosilactobacillus spp. bacteria that are beneficial to the user and may have antagonistic activity against deleterious, pathogenic bacteria. Suitable postbiotic ferments include, but are not limited to, peptidoglycan, teichoic acid, S-layer proteins, short-chain fatty acids, conjugated linoleic acid, exopolysacchardies, bacteriocins, lanthipeptides, and D / L-lactic acid. Further, in certain example embodiments, Limosilactobacillus vaginalis bacteria can be utilized alone or in combination with Lactobacillus spp. bacteria to create a postbiotic ferment.

[0050] Without intending to be limited by theory, the present inventors have found that microbiome modulating compositions which include only a postbiotic ferment of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof may have a therapeutic effect in promoting the growth of healthy Lactobacillus spp. bacteria while inhibiting the growth of deleterious, pathogenic bacteria. For instance, in some example embodiments, when the microbiome modulating composition is applied to a culture of L. crispatus on Minimal Lactobacillus Media (MLM) culture media, the culture of L crispatus demonstrates a lag time in growth of about 105 minutes or less. In other example embodiments, when the microbiome modulating composition is applied to a culture of L. crispatus on Minimal Lactobacillus Media (MLM) culture media, the culture of L. crispatus demonstrates a growth rate during exponential growth of about 0.004 AOD / min or greater. In certain example embodiments, when the microbiome modulating composition is applied to a culture of L. crispatus on Minimal Lactobacillus Media (MLM) culture media, a final bacterial concentration (OD at 600 nm) of L. crispatus is about 1 .55 or greater. Thus, the presence of the postbiotic ferment in the microbiome modulating composition may improve the growth kinetics of L. crispatus, as it allows for a reduction in lag time, a faster doubling rate during the exponential growth phase, and a higher stationary phase final bacteria composition in comparison to growth of L crispatus alone or when the pH of the media was lowered to match the pH of the extract.

[0051] Likewise, the presence of only a postbiotic ferment of at least two of any Lactobacillus spp. bacteria, Limosilactobaciilus spp. bacteria, or combinations thereof may inhibit the growth of deleterious, pathogenic bacteria, such as Enterococcus faecalis. For instance, in certain example embodiments, when the microbiome modulating composition is applied to a culture of E. faecalis on Minimal Lactobacillus Media (MLM) culture media, the culture of E. faecalis demonstrates a growth rate during exponential growth of about 0.0006 AOD / min or less. In other example embodiments, when the microbiome modulating composition is applied to a culture of E. faecalis on Minimal Lactobacillus Media (MLM), the final bacterial concentration (OD at 600 nm) of E. faecalis is about 0.3 or less.

[0052] However, other therapeutic agents may be added to the composition to improve the efficacy of the postbiotic ferments. Suitable additional therapeutic agents often synergistically interact with the postbiotic ferments to enhance the growth of healthy lactobacilli and thus, re-establish healthy vaginal microflora. For instance, in some example embodiments, a probiotic is added to the microbiome modulating composition including a postbiotic ferment. Probiotics, for instance, are live microorganisms, such as bacteria and yeast, that are intended to maintain or improve the healthy bacteria in the body. In some example embodiment, for instance, suitable probiotics to be added to the composition to quickly reestablish a healthy vaginal environment include, but are not limited to, a mixture of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillus johnsonil, Limosilactobaciilus vaginalis, Limosilactobaciilus reuteri, Limosilactobaciilus fermentum, or Limosilactobaciilus rhamnosus. In preferred embodiments, a mixture of two or more probiotics are added to the composition. For instance, in a preferred embodiment, a probiotic including a mixture of Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus iners may be added to the microbiome modulating composition. Without intending to be limited by theory, the present inventors have found enhanced results when a microbiome modulating composition including both a postbiotic ferment and a probiotic, as described above, are used in combination. For instance, in some example embodiments, when the microbiome modulating composition including both a postbiotic ferment and a probiotic is applied to a culture of L crispatus on Minimal Lactobacillus Media (MLM) culture media, the culture of L. crispatus demonstrates a lag time in growth of about 100 minutes or less, such as about 95 minutes or less, such as about 90 minutes or less, such as about 80 minutes or less. In other example embodiments, when the microbiome modulating composition including both a postbiotic ferment and a probiotic is applied to a culture of L. crispatus on Minimal Lactobacillus Media (MLM) culture media, the culture of t. crispatus demonstrates a growth rate during exponential growth of about 0.0045 AOD / min or greater, such as about 0.00475 AOD / min or greater, such as about 0.005 AOD / min or greater, such as about 0.0051 AOD / min or greater. In certain example embodiments, when the microbiome modulating composition including both a postbiotic ferment and a probiotic is applied to a culture of L. crispatus on Minimal Lactobacillus Media (MLM) culture media, a final bacterial concentration (OD at 600 nm) of L. crispatus is about 1 .60 or greater, such as about 1 .65 or greater, such as about 1 .70 or greater.

[0053] Further, the combination of the postbiotic ferment of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof in combination with a probiotic including a mixture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof may inhibit the growth of deleterious, pathogenic bacteria, such as Enterococcus faecalis. For instance, in certain example embodiments, when the microbiome modulating composition including a postbiotic ferment and a probiotic is applied to a culture of E. faecalis on Minimal Lactobacillus Media (MLM) culture media, the culture of E. faecalis demonstrates a growth rate during exponential growth of about 0.0005 AOD / min or less, such as 0.0004 AOD / min or less, such as 0.0003 AOD / min or less. In other example embodiments, when the microbiome modulating composition including a postbiotic ferment and a probiotic is applied to a culture of E. faecalis on Minimal Lactobacillus Media (MLM) culture media, the final bacterial concentration (OD at 600 nm) of E. Faecalis is about 0.3 or less, such as about 0.25 or less, such as about 0.2 or less, such as about 0.16 or less.

[0054] In other example embodiments, the microbiome modulating composition may also include one or more additional prebiotics. Prebiotics differ from probiotics because probiotics encompass the bacteria themselves, whereas prebiotics are merely food sources for the bacteria. Therefore, while probiotics include living microorganisms, prebiotics do not. Particularly suitable prebiotics include, but are not limited to, disaccharides (i.e., lactulose), fructans, fructooligosaccharides ( / .e., kestose), galactooligosaccharides, glucans, glucooligosaccharides, isomaltooligosaccharides ( / .e., isomaltopentaose, isomaltose, isomaltotetraose, isomaltotriose, isomaltulose, isopanose, kojibiose, nigerose, panose), oligosaccharides ( / .e., maltodextrin), polysaccharides / polysaccharide polymers ( / .e., amylopectin, amylose, dextran, dextrin, polydextrose, pullulan), sugar alcohols ( / .e., lactitol), tetrasaccharides ( / .e., stachyose), and trisaccharides ( / .e., lactosucrose, lactulosucrose, raffinose) or combinations thereof. In preferred embodiments, the prebiotic includes at least one Lactobacillus spp. bacteria-specific prebiotic, at least one Limosilactobacillus spp. bacteria-specific prebiotic, or combinations thereof.

[0055] It should be understood by one of ordinary skill in the art that the therapeutic agent of the microbiome modulating composition may include a postbiotic ferment, a probiotic, a prebiotic, or combinations thereof. Accordingly, in other example embodiments, compositions of the present disclosure comprise less than about 10% wt / vol of the postbiotic ferment and any other prebiotic or probiotic compounds added to the composition. In some example embodiments the total amount of the postbiotic ferment, probiotic, and / or prebiotic is less than about 7.5% wt / vol, or less than about 5% wt / vol, such as from about 0.01 % to about 4% wt / vol, or from about 0.1 % to about 2% wt / vol. For instance, in some example embodiments, the postbiotic ferment, the probiotic, and any optional prebiotics comprise about 0.1% wt / vol to about 10% wt / vol of the microbiome modulating composition.

[0056] The present disclosure is also generally directed towards a method for treating microbial dysbiosis of an urogenital area or a skin area of a user. The method comprises applying a microbiome modulating composition, as discussed above, to an urogenital or a skin area of a user. In certain example embodiments, the method further includes promoting a healthy microflora balance of the urogenital area or the skin area of the user, wherein promoting the healthy microflora balance of the urogenital area or the skin area of the user comprises promoting a growth of Lactobacillus spp. bacteria relative to a growth of one or more types of Enterococcus spp. bacteria, Staphylococcus spp. bacteria, Escherichia spp. bacteria, Gardnerella spp. bacteria, Streptococcus spp. bacteria, or mixtures thereof. In certain example embodiments, the ratio of the growth of the Lactobacillus spp. bacteria to the deleterious, pathogenic bacteria is from about 4 to about 12, such as about 5 to about 11 , such as about 5.5 to about 10.5.

[0057] Accordingly, the composition can be applied to any urogenital surface or skin surface to promote growth of healthy bacteria and to modulate a balanced microbiome. In certain embodiments, the methods and compositions disclosed herein can improve overall skin health and maintenance of the skin barrier by increasing production of lactic acid, and can be applied to any skin surface including, but not limited to, epithelium, mucous membranes, and any other tissue forming the outer layer of a body's surface. In preferred embodiments, the methods and compositions can improve urogenital health, and can be applied to any urogenital surface including, but not limited to, the vaginal canal, vulva, vagina, urethra, urinary tract, bladder, and surrounding areas. Such methods and compounds may be considered for the maintenance of vaginal and bladder health, the promotion of balanced intimate skin, the promotion of urinary tract health, and the prevention of bacterial vaginosis, yeast infections, urinary tract infections, incontinence, overactive bladder, pre-term labor, infertility, and other bacterial infections relevant to intimate wellness. In other embodiments, such methods and compounds disclosed herein may be associated with cosmetic endpoints including healthy discharge, less itching, and reduction of vaginal malodor.

[0058] The microbiome modulating compositions of the present disclosure can be administered in several forms to a user. For instance, in certain embodiments, the microbiome modulating composition may be applied to a substrate, the substrate comprising a wipe or at least a portion of an absorbent article. For example, compositions may be prepared as formulations for administration to a user or may be applied to a substrate, such as a wiping substrate, for administration to a user. Preferably the postbiotic ferments, the probiotics, the prebiotics, or a combination thereof are useful in the present disclosure are soluble to facilitate their formulation for administration to a user.

[0059] In certain example embodiments, the compositions can be configured to be administered to a user through topical application in various forms, including, but not limited to, a liquid, cream, gel, spray, or suppository. In further example embodiments, the composition can be formulated as a treatment formulation, such that it may be formulated as a moisturizer, lotion, jelly, liniment, ointment, salve, oil, foam, film, wash, slow-releasing polymer, coating, liquid, vaginal capsule, vaginal tablet, vaginal film, vaginal sponge, vaginal ovule, etc. The composition may also be applied to a vaginal insert, tampon, wipe or pad, and then administered to the vagina. Thus, in preferred example embodiments, the microbiome modulating composition is a vaginal insertable gel. The vaginal insertable gel may be applied directly to the vaginal canal or may applied to an insertable substrate, such as to a tampon, a menstrual cup, a menstrual sponge, a suppository, a pad, a single use pessary, and / or a reusable pessary. Without intending to be limited by theory, the present inventors have found that by utilizing a postbiotic ferment from a mixture of Lactobacillus spp. bacteria, either alone or in combination with a probiotic mixture of Lactobacillus spp. bacteria, may mimic or “re-create” a healthy vaginal microenvironment that is inhospitable to infectious microbes, as healthy vaginal microenvironments include high concentrations of lactobacilli.

[0060] In other embodiments, the composition may include other components such as, for example, an additional prebiotic, a surfactant, an ester, a humectant, a pH adjuster, a rheology modifier, a gelling agent, a nitrogen source, and an antimicrobial agent.

[0061] Additional Prebiotics

[0062] In some embodiments, the composition can include one or more additional prebiotics that are different than the at least one Lactobacillus spp. bacteria-specific prebiotic, the at least one Limosilactobacillus spp. bacteria-specific prebiotic, or combinations thereof, the present disclosure. The additional prebiotics may have a synergistic effect in combination with the at least one Lactobacillus spp. bacteria-specific prebiotic, the at least one Limosilactobacillus spp. bacteria-specific prebiotic, or combinations thereof. Suitable additional prebiotics can be selected from a group comprising a-D- lactose, maltitol, N-acetylglucosamine, o-cyclodextrin, [3-cyclodextrin, glucomannan, D-tagatose, 2- deoxy-D-ribose, o-methyl-D-glucoside, amylopectin, |3-D-fructose, [3-D-glucose, D-arabinose, D- cellobiose, dextran, dextrin type I, dextrin type II, malate, D-trehalose, 4-O-P-D-galactopyranosyl-D- glucitol, lactitol, lactulose, maltotriose, isomaltulose, pectin, pullulan, and salicin.

[0063] Surfactant

[0064] In some embodiments, the microbiome modulating composition can include one or more surfactants. In an embodiment where the composition is included in a wipe, the composition may also likely include one or more surfactants. These may be selected from anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants. Amounts of surfactants may range from 0.01 % to 30%, or from 10% to 30%, or from 0.05% to 20%, or from 0.10% to 15% by total weight of the composition. In some embodiments, such as when the wetting composition is used with a wipe, the surfactant can comprise less than 5% by total weight of the wetting composition.

[0065] Suitable anionic surfactants include, but are not limited to, C8 to C22 alkane sulfates, ether sulfates and sulfonates. Among the suitable sulfonates are primary C8 to C22 alkane sulfonate, primary C8 to C22 alkane disulfonate, C8 to C22 alkene sulfonate, C8 to C22 hydroxyalkane sulfonate or alkyl glyceryl ether sulfonate. Specific examples of anionic surfactants include ammonium lauryl sulfate, ammonium laureth sulfate, triethylamine lauryl sulfate, triethylamine laureth sulfate, triethanolamine lauryl sulfate, triethanolamine laureth sulfate, monoethanolamine lauryl sulfate, monoethanolamine laureth sulfate, diethanolamine lauryl sulfate, diethanolamine laureth sulfate, lauric monoglyceride sodium sulfate, sodium lauryl sulfate, sodium laureth sulfate, potassium laureth sulfate, sodium lauryl sarcosinate, sodium lauroyl sarcosinate, potassium lauryl sulfate, sodium trideceth sulfate, sodium methyl lauroyl taurate, sodium lauroyl isethionate, sodium laureth sulfosuccinate, sodium lauroyl sulfosuccinate, sodium tridecyl benzene sulfonate, sodium dodecyl benzene sulfonate, sodium lauryl amphoacetate and mixtures thereof. Other anionic surfactants include the C8 to C22 acyl glycinate salts. Suitable glycinate salts include sodium cocoylglycinate, potassium cocoylglycinate, sodium lauroylglycinate, potassium lauroylglycinate, sodium myristoylglycinate, potassium myristoylglycinate, sodium palmitoylglycinate, potassium palmitoylglycinate, sodium stearoylglycinate, potassium stearoylglycinate, ammonium cocoylglycinate and mixtures thereof. Cationic counter-ions to form the salt of the glycinate may be selected from sodium, potassium, ammonium, alkanolammonium and mixtures of these cations.

[0066] Suitable cationic surfactants include, but are not limited to alkyl dimethylamines, alkyl amidopropylamines, alkyl imidazoline derivatives, quaternised amine ethoxylates, and quaternary ammonium compounds.

[0067] Suitable nonionic surfactants include, but are not limited to, alcohols, acids, amides or alkyl phenols reacted with alkylene oxides, especially ethylene oxide either alone or with propylene oxide. Specific nonionics are C6 to C22 alkyl phenols-ethylene oxide condensates, the condensation products of C8 to C13 aliphatic primary or secondary linear or branched alcohols with ethylene oxide, and products made by condensation of ethylene oxide with the reaction products of propylene oxide and ethylenediamine. Other nonionics include long chain tertiary amine oxides, long chain tertiary phosphine oxides and dialkyl sulphoxides, alkyl polysaccharides, amine oxides, block copolymers, castor oil ethoxylates, ceto-oleyl alcohol ethoxylates, ceto-stearyl alcohol ethoxylates, decyl alcohol ethoxylates, dinonyl phenol ethoxylates, dodecyl phenol ethoxylates, end-capped ethoxylates, ether amine derivatives, ethoxylated alkanolamides, ethylene glycol esters, fatty acid alkanolamides, fatty alcohol alkoxylates, lauryl alcohol ethoxylates, mono-branched alcohol ethoxylates, natural alcohol ethoxylates, nonyl phenol ethoxylates, octyl phenol ethoxylates, oleyl amine ethoxylates, random copolymer alkoxylates, sorbitan ester ethoxylates, stearic acid ethoxylates, stearyl amine ethoxylates, synthetic alcohol ethoxylates, tall oil fatty acid ethoxylates, tallow amine ethoxylates and trid tridecanol ethoxylates.

[0068] Suitable zwitterionic surfactants include, for example, alkyl amine oxides, alkyl hydroxysultaines, silicone amine oxides, and combinations thereof. Specific examples of suitable zwitterionic surfactants include, for example, 4-[N,N-di(2-hydroxyethyl)-N-octadecylammonio]-butane-1- carboxylate, S-[S-3-hydroxypropyl-S-hexadecylsulfonio]-3-hydroxypentane-1-sulfate, 3-[P,P-diethyl-P- 3, 6, 9-trioxatetradexopcylphosphonio]-2-hydroxypropane-1 -phosphate, 3-[N,N-dipropyl-N-3-dodecoxy-2- hydroxypropylammonio]-propane-1 -phosphonate, 3-(N,N-dimethyl-N-hexadecylammonio)propane-1- sulfonate, 3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxypropane-1-sulfonate, 4-[N,N-di(2- hydroxyethyl)-N-(2-hydroxydodecyl)ammonio]-butane-1-carboxylate, 3-[S-ethyl-S-(3-dodecoxy-2- hydroxypropyl)sulfonio]-propane-1 -phosphate, 3-[P,P-dimethyl-P-dodecylphosphonio]-propane-1- phosphonate, 5-[N,N-di(3-hydroxypropyl)-N-hexadecylammonio]-2-hydroxy-pentane-1 -sulfate, lauryl hydroxysultaine and combinations thereof.

[0069] Suitable amphoteric surfactants include, but are not limited to, derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds, in which the aliphatic radicals can be straight or branched chain, and wherein one of the aliphatic substituents contains from about 8 to about 18 carbon atoms and one substituent contains an anionic group (e.g., carboxy, sulfonate, sulfate, phosphate, or phosphonate). Illustrative amnphoterics are coco dimethyl carboxymethyl betaine, cocoamidopropyl betaine, cocobetaine, oleyl betaine, cetyl dimethyl carboxymethyl betaine, lauryl bis- (2-hydroxyethyl) carboxymethyl betaine, stearyl bis-(2-hydroxypropyl) carboxymethyl betaine, oleyl dimethyl gamma-carboxypropyl betaine, lauryl bis-(2-hydroxypropyl)alpha-carboxyethyl betaine, cocoamphoacetates, and combinations thereof. The sulfobetaines may include stearyl dimethyl sulfopropyl betaine, lauryl dimethyl sulfoethyl betaine, lauryl bis-(2-hydroxyethyl) sulfopropyl betaine and combinations thereof.

[0070] Esters

[0071] In some embodiments, the microbiome modulating compositions include one or more esters. The esters may be selected from cetyl palmitate, stearyl palmitate, cetyl stearate, isopropyl laurate, isopropyl myristate, isopropyl palmitate, and combinations thereof. The fatty alcohols include octyldodecanol, lauryl, myristyl, cetyl, stearyl, behenyl alcohol, and combinations thereof. The fatty acids can include, but are not limited to, capric acid, undecylenic acid, lauric acid, Myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidic acid, and behenic acid. Ethers such as eucalyptol, ceteraryl glucoside, dimethyl isosorbic polyglyceryl-3 cetyl ether, polyglyceryl-3 decyltetradecanol, propylene glycol myristyl ether, and combinations thereof can also suitably be used as emollients. Other suitable ester compounds for use in the antimicrobial compositions or the present disclosure are listed in the International Cosmetic Ingredient Dictionary and Handbook, 11th Edition, CTFA, (January, 2006) ISBN-10: 1882621360, ISBN-13: 978-1882621361 , and in the 2007 Cosmetic Bench Reference, Allured Pub. Corporation (July 15, 2007) ISBN-10: 1932633278, ISBN-13: 978-1932633276, both of which are incorporated by reference herein to the extent they are consistent herewith. Humectants

[0072] In some embodiments, the microbiome modulating compositions include one or more humectants. Humectants that are suitable as carriers in the compositions of the present disclosure include, for example, glycerin, glycerin derivatives, hyaluronic acid, hyaluronic acid derivatives, betaine, betaine derivatives, amino acids, amino acid derivatives, glycosaminoglycans, glycols, polyols, sugars, sugar alcohols, hydrogenated starch hydrolysates, hydroxy acids, hydroxy acid derivatives, salts of PCA and the like, and combinations thereof. Specific examples of suitable humectants include honey, sorbitol, hyaluronic acid, sodium hyaluronate, betaine, lactic acid, citric acid, sodium citrate, glycolic acid, sodium glycolate .sodium lactate, urea, propylene glycol, butylene glycol, pentylene glycol, ethoxydiglycol, methyl gluceth-10, methyl gluceth-20, polyethylene glycols (as listed in the International Cosmetic Ingredient Dictionary and Handbook such as PEG-2 through PEG 10), propanediol, xylitol, maltitol, or combinations thereof.

[0073] The compositions of the disclosure may include one or more humectants in an amount of about 0.01% (by total weight of the composition) to about 20% (by total weight of the composition), or about 0.05% (by total weight of the composition) to about 10% by total weight of the composition), or about 0.1 % (by total weight of the composition) to about 5.0% (by total weight of the composition). pH adjusting agent

[0074] In some embodiments, the microbiome modulating compositions of the present disclosure can be acidic, i.e. , have a pH less than about 7.0 and more preferably less than about 6.0, such as from about 3.0 to about 6.0 and still more preferably from about 3.8 to about 5.0. In a particularly preferred embodiment, the pH may be maintained at a mildly acidic level to correspond to normal vaginal conditions, the environment in which the composition will typically be delivered. For example, the pH may be within a range of from about 3.0 to about 6 0, in some embodiments from about 3.5 to about 5.0, and in some embodiments, from about 4.0 to about 4.5. The foregoing acid pH may also provide other benefits. For instance, when the composition is configured to form a gel, such as described below, a low pH level may also improve the gelation rate and gel strength to reduce the likelihood of leakage just after insertion of the composition into the vagina.

[0075] The pH of the composition may be adjusted using an organic acid. Organic acids useful in the present disclosure generally consist of mono- or polycarboxylic acids having one or more hydroxyl functional groups at least one of which is introduced into the a-position (i.e., on the carbon atom adjacent to the carboxyl functional group). Examples of particularly useful organic acids can include citric acid, lactic acid, methyllactic acid, phenyllactic acid, malic acid, mandelic acid, glycolic acid, tartronic acid, tartaric acid and gluconic acid. In particularly preferred embodiments the organic acid is selected from the group consisting of citric acid, lactic acid, malic acid, glycolic acid, and tartaric acid. In certain embodiments the organic acid may be provided with an appropriate counterion, such as calcium, sodium, or magnesium. In other example embodiments, the pH adjusting agent includes D / L- Lactic acid, maleic acid, citric acid, benzoic acid, sodium benzoate, and / or sodium acetate.

[0076] In view of the foregoing, in certain embodiments the compositions and formulations of the present disclosure may be buffered to have a pH from about 3.0 to about 6.0, more preferably from about 3.8 to about 5.0.

[0077] Rheology modifier

[0078] Optionally, one or more rheology modifiers, such as thickeners, may be added to the microbiome modulating composition. Suitable rheology modifiers are compatible with the postbiotic ferments. As used herein, "compatible” refers to a compound that, when mixed with the postbiotic ferments, either alone or in further combination with a prebiotic or a probiotic, does not adversely affect the properties of the postbiotic ferments.

[0079] A thickening system is used in the compositions to adjust the viscosity and stability of the compositions. Specifically, thickening systems prevent the composition from running off of the hands or body during dispensing and use of the composition. When the composition is used with a wipe product, a thicker formulation can be used to prevent the composition from migrating from the wipe substrate.

[0080] The thickening system should be compatible with the compounds used in the present disclosure; that is, the thickening system, when used in combination with the therapeutic agent, should not precipitate out, form a coacervate, or prevent a user from perceiving the conditioning benefit (or other desired benefit) to be gained from the composition. The thickening system may include a thickener which can provide both the thickening effect desired from the thickening system and a conditioning effect to the user.

[0081] Thickeners may include cellulosics, gums, acrylates, starches, and various polymers. Suitable examples include but are not limited to hydroxethyl cellulose, xanthan gum, guar gum, potato starch, and corn starch. In some embodiments, PEG-150 stearate, PEG-150 distearate, PEG-175 diisostearate, polyglyceryl-10 behenate / eicosadioate, disteareth-100 IPDI, polyacrylamidomethylpropane sulfonic acid, butylated PVP, and combinations thereof may be suitable.

[0082] While the viscosity of the compositions will typically depend on the thickener used and the other components of the compositions, the thickeners of the compositions suitably provide for a composition having a viscosity in the range of greater than 1 cP to about 30,000 cP or more. In another embodiment, the thickeners provide compositions having a viscosity of from about 100 cP to about 20,000 cP. In yet another embodiment, thickeners provide compositions having a viscosity of from about 200 cP to about 15,000 cP. In embodiments where the compositions are included in a wipe, the viscosity may range from about 1 cP to about 2000 cP. In some embodiments, it is preferable to have a viscosity of the composition be less than 500 cP.

[0083] When including a thickening system, the compositions of the present disclosure can include the thickening system in an amount of no more than about 20% (by total weight of the composition), or from about 0.01% (by total weight of the composition) to about 20% (by total weight of the composition). In another aspect the thickening system is present in the antimicrobial composition in an amount of from about 0.10% (by total weight of the composition) to about 10% (by total weight of the composition), or from about 0.25% (by total weight of the composition) to about 5% (by total weight of the composition), or from about 0.5% (by total weight of the composition) to about 2% (by total weight of the composition).

[0084] In one embodiment, the compositions may include hydrophobic and hydrophilic ingredients, such as a lotion or cream. Generally, these emulsions have a dispersed phase and a continuous phase and are generally formed with the addition of a surfactant or a combination of surfactants with varying hydrophilic / lipophilic balances (HLB). Suitable emulsifiers include surfactants having HLB values from 0 to 20, or from 2 to 18. Suitable non-limiting examples include Ceteareth-20, Cetearyl Glucoside, Ceteth-10, Ceteth-2, Ceteth-20, Cocamide MEA, Glyceryl Laurate, Glyceryl Stearate, PEG-100 Stearate, Glyceryl Stearate, Glyceryl Stearate SE, Glycol Distearate, Glycol Stearate, lsosteareth-20, Laureth-23, Laureth-4, Lecithin, , Methyl Glucose Sesquistearate, Oleth-10, Oleth-2, Oleth-20, PEG- 100 Stearate, PEG-20 Almond Glycerides, PEG-20 Methyl Glucose Sesquistearate, PEG-25 Hydrogenated Castor Oil, PEG-30 Dipolyhydroxystearate, PEG-4 Dilaurate, PEG-40 Sorbitan Peroleate, PEG-60 Almond Glycerides, PEG-7 Olivate, PEG-7 Glyceryl Cocoate, PEG-8 Dioleate, PEG-8 Laurate, PEG-8 Oleate, PEG-80 Sorbitan Laurate, Polysorbate 20, Polysorbate 60, Polysorbate 80, Polysorbate 85, Propylene Glycol Isostearate, Sorbitan Isostearate, Sorbitan Laurate, Sorbitan Monostearate, Sorbitan Oleate, Sorbitan Sesquioleate, Sorbitan Stearate, Sorbitan Trioleate, Stearamide MEA, Steareth-100, Steareth-2, Steareth-20, Steareth-21. The compositions can further include surfactants or combinations of surfactants that create liquid crystalline networks or liposomal networks. Suitable non-limiting examples include OLIVEM 1000 (INCI: Cetearyl Olivate (and) Sorbitan Olivate (available from HallStar Company (Chicago, IL)); ARLACEL LC (INCI: Sorbitan Stearate (and) Sorbityl Laurate, commercially available from Croda (Edison, NJ)); CRYSTALCAST MM (INCI: Beta Sitosterol (and) Sucrose Stearate (and) Sucrose Distearate (and) Cetyl Alcohol (and) Stearyl Alcohol, commercially available from MMP Inc. (South Plainfield, NJ)); UNIOX CRISTAL (INCI: Cetearyl Alcohol (and) Polysorbate 60 (and) Cetearyl Glucoside, commercially available from Chemyunion (Sao Paulo, Brazil)). Other suitable emulsifiers include lecithin, hydrogenated lecithin, lysolecithin, phosphatidylcholine, phospholipids, and combinations thereof.

[0085] Gelling agents

[0086] In preferred example embodiments, the microbiome modulating composition is in the form of a vaginally insertable gel. In particularly suitable example embodiments, the gel serves as a mucoadhesive component. Particularly suitable mucoadhesive components include Carbopol® 980 (Cb) (Lubrizol, USA), Gelcarin® GP-812 NF (Cg) (PMC BioPolymer, USA), Pemulen™ TR-1 (C) (Lubrizol, USA), guar gum (G) (Chopra Gums, India), glycogen, and xanthan gum. In certain example embodiments, the mucoadhesive further comprises other excipients, such as propylene glycol, methylparaben, sorbic acid, sodium hydroxide or hydrochloride acid and deionized water. In some example embodiments, the mucoadhesive also may include hydroxyethylcellulose (Natrosol 250 HHX Pharm), Pluronic®-based multiblock copolymers, poloxamer, and hyaluronic acid.

[0087] In certain example embodiments, the disperse phase of the gel may be formed from any of a variety of different gelling agents, including temperature responsive (“thermogelling”) compounds, ion responsive compounds, and so forth. Thermogelling systems, for instance, respond to a change in temperature (e.g., increase in temperature) by changing from a liquid to a gel. Generally speaking, the temperature range of interest is from about 25°C to about 40°C, in some embodiments from about 35°C to about 39°C, and in one particular embodiment, at the human body temperature (about 37°C). In some cases, thermogelling block copolymers, graft copolymers, and / or homopolymers may be employed. For example, polyoxyalkylene block copolymers may be used in some embodiments of the present invention to form a thermo-gelling composition. Suitable thermo-gelling compositions may include, for example, homopolymers, such as poly(N-methyl-N-n-propylacrylamide), poly(N-n- propylacrylamide), poly(N-methyl-N-isopropylacrylamide), poly(N-n-propylmethacrylamide), poly(N- isopropylacrylamide), poly(N,n-diethylacrylamide); poly(N-isopropylmethacrylamide), poly(N- cyclopropylacrylamide), poly(N-ethylmethyacrylamide), poly(N-methyl-N-ethylacrylamide), poly(N- cyclopropylmethacrylamide), and poly(N-ethylacrylamide). Still other examples of suitable thermogelling polymers may include cellulose ether derivatives, such as hydroxypropyl cellulose, methyl cellulose, hydroxypropylmethyl cellulose, and ethylhydroxyethyl cellulose. Moreover, thermogelling polymers may be made by preparing copolymers between (among) monomers, or by combining such homopolymers with other water-soluble polymers, such as acrylic monomers (e.g., acrylic or methacrylic acid, acrylate or methacrylate, acrylamide or methacrylamide, and derivatives thereof). In one particular embodiment of the present disclosure, for example, the composition is configured to rapidly form a gel when applied to the vagina. A ''gel” is a colloid in which a disperse phase combines with a dispersion medium to produce a jelly-like, solid, or semi-solid material. The gel may form in less than about one hour, in some embodiments less than about one minute, and in some embodiments, less than about 30 seconds. Among other things, such rapid gelation reduces the likelihood of leakage during use. In addition, because the gel may form I ntravag inally , it is more likely to retain its structure and shape over an extended period of time. In this manner, the gel may provide the prolonged release of a therapeutic agent that inhibits and / or treats vaginal infection. For instance, the gel may remain within the vagina for about 2 to about 48 hours to provide the desired effect to modulate the urogenital microbiome of the user.

[0088] Although a variety of compounds may be employed, water is usually employed as the dispersion medium for the gel to optimize biocompatibility. Other possible dispersion mediums include non-aqueous solvents, including glycols, such as propylene glycol, butylene glycol, triethylene glycol, hexylene glycol, polyethylene glycols, ethoxydiglycol, and dipropyleneglycol; alcohols, such as ethanol, n-propanol, and isopropanol; triglycerides; ethyl acetate; acetone; triacetin; and combinations thereof. Typically, the dispersion medium (e.g., water) constitutes greater than about 75% wt / vol, in some embodiments greater than about 90% wt / vol, and in some embodiments, from about 95% to about 99% wt / vol of the composition.

[0089] The compositions of the present disclosure may also include an ion responsive compound. Such compounds are generally well known in the art and tend to form a gel in the presence of certain ions or at a certain pH. For instance, one suitable class of ion responsive compounds that may be employed in the present disclosure is anionic polysaccharides. Anionic polysaccharides may form a three-dimensional polymer network that functions as the disperse phase of the gel. Generally speaking, anionic polysaccharides include polysaccharides having an overall anionic charge, as well as neutral polysaccharides that contain anionic functional groups.

[0090] Any of a variety of anionic polysaccharides capable of forming a gel when contacted with vaginal mucosa may be used in the present disclosure. Such gel-forming anionic polysaccharides are typically stable over the normal acidic pH values found in the vagina (e.g., from about 2.5 to about 5.5). For instance, some suitable examples of gel-forming anionic polysaccharides include natural gums, such as gellan gum and alginate gums (e.g., ammonium and alkali metal of salts of alginic acid); chitosan; carboxymethylcellulose, pectins, carrageenan, xantham gum, and derivatives or salts thereof. The particular type of anionic polysaccharide selected will depend, in part, on the nature of the composition and the other components used therein. For example, carrageenan is sensitive to particular types of cations, e.g., it typically gels in the presence of potassium but not sodium. Glycuronans, likewise, typically gel in the presence of divalent cations (e.g., Ca2+), but not monovalent cations (e.g., Na+). Xanthan gum may gel in the presence of divalent cations, but only at a relatively high pH.

[0091] Although any of the above-described anionic polysaccharides may be used in the present disclosure, gellan gum is particularly desired for use in the present disclosure, either alone or in combination with other gelling agents, because it is able to form a gel in the presence of a wide variety of different cations, including both monovalent and divalent cations. Gellan gum is intended to encompass any form of gellan, including native gellan, clarified gellan, deacylated gellan, nonacylated gellan (e.g., produced from genetically engineered bacteria), clarified gellan (the polysaccharide is fully or partially removed from the bacterial debris), chemically modified gellan, etc. Various types of gellan gums and methods for forming such gums are described in U.S. Pat. Nos. 4,326,052, 4,326,053, 4,377,636, 4,385,123, and 4,563,366. Suitable gellan gums are commercially available from a variety of different sources. For example, GELRITE™ gellan gum is available from Sigma-Aldrich Chemical Co. of St. Louis, MO, and is produced from a naturally occurring polysaccharide after deacylation and clarification. Deacylated gellan is also available from CP Kelco U.S., Inc. of Chicago, IL under the name KELCOGEL®.

[0092] Gellan gum may be either high or low acyl gellan. In the high acyl (or “native”) form, two acyl substituents, acetate and glycerate, are present. Both substituents are located on the same glucose residue and, on average, there is one glycerate per repeat unit and one acetate per every two repeat units. In the low acyl form, the acyl groups may be wholly or partially removed through deacylation. The degree of deacylation of deacylated gellan gums may be at least about 20%, in some embodiments at least about 50%, and in some embodiments, at least about 75%. Alternatively, the low acyl gellan gum may simply be “nonacylated” in that it is formed without acyl groups by genetically engineered bacteria. Regardless of the manner in which they are formed, low acyl gellan gums generally have a gelation temperature within the range 30 to 50°C, which may be particularly well suited for use in the present disclosure so that it may gel at body temperatures of about 37°C, but remain stable at typical storage and transportation temperatures of about 25°C. In addition, low acyl gellan gums are also firm and elastic, and thus may retain their shape after delivery to the vaginal cavity.

[0093] In most embodiments the gelling agent(s) are present in an amount of from about 0.01% to about 10.0% wt / vol, in some embodiments from about 0.05% to about 5.0% wt / vol, and in some embodiments, from about 0.1% to about 1 .0% wt / vol of the composition. If desired, a gelling composition may be provided in any desired form (e.g., liquid, powder, etc.). In fact, one particular benefit of the composition is that it may be administered as a liquid, which allows for the selection of a wider variety of administration techniques than would otherwise be available for a solid or semi-solid gel. One technique that may be employed includes dispensing the composition through a liquid applicator, such as a syringe or tube, into the vaginal cavity. The administered volume of the composition may constitute a single dose or two or more doses. Although not necessarily required, the composition of may also be sterilized prior to administration. Sterilization may be accomplished by any technique known in the art, such as using a gas (e.g., ethylene oxide), radiation (e.g., gamma), or heat (autoclaving). If desired, the composition may be subjected to one or more filtration steps prior to sterilization to help remove contaminants.

[0094] Nitrogen Sources

[0095] In some embodiments, the microbiome modulating compositions may include one or more nitrogen sources to drive potential synergies. Some suitable nitrogen sources include nitrogen heterocycles, ammonia, ammonium, urea, amino acids, and all salts of ammonium compounds, for example chloride, carbonate, sulfate, bicarbonate, nitrate, acetate, and molybdate. Suitable amino acids include, but are not limited to, slowly assimilated amino acids such as proline, which are often included to promote fermentation and encourage high productivity of secondary metabolites. Other suitable nitrogen sources include alloxan, ammonium citrate, glycine, L-cysteine, L-glutamic acid, L- glutamine, L-homoserine, L-leucine, L-lysine, L-methionine, L-tyrosine, cytidine, D-asparagine, adenosine, H-Ala-Thr-OH, D-glucuronamide, Ala-asp, Ala-His, Gly-Met, N-acetyl-D-galactosamine, pyrimidine, L-serine, inosine, D-alanine, N-acetyl-D-mannosamine, Gly-Asn, Ala-Glu, D-galactosamine HOI, D-glucosamine HCI, DL-a-amino-n-butyric acid, D-mannosamine HOI, Gly-GIn, Gly-Glu, DL- lactamide, Met-Ala, Methyl(2-phenylethyl)amine hydrochloride, Glycyl-alanine, DL-y-amino-n-butyric acid, and N-acetyl-D-glucosamine.

[0096] Antimicrobial agents

[0097] In some embodiments, the microbiome modulating composition may include one or more antimicrobial agents to increase shelf life. Some suitable antimicrobial agents that may be used in the present disclosure include traditional antimicrobial agents. As used herein, "traditional antimicrobial agents” means compounds that have been historically recognized by regulatory bodies as providing an antimicrobial effect, such as those listed in the European Union’s Annex V list of preservatives allowed in cosmetics products. Traditional antimicrobial agents include, but are not limited to: succinic acid and salts thereof, propionic acid and salts thereof; salicylic acid and salts thereof; sorbic acid and salts thereof; benzoic acid and salts and esters thereof; formaldehyde; paraformaldehyde; o-phenylphenol and salts thereof; zinc pyrithione; inorganic sulfites; hydrogen sulfites; chlorobutanol; benzoic parabens, such as methylparaben, propylparaben, butylparaben, ethylparaben, isopropylparaben, isobutylparaben, benzylparaben, sodium methylparaben and sodium propylparaben; dehydroacetic acid and salts thereof; formic acid and salts thereof; dibromohexamidine isethionate; thimerosal; phenylmercuric salts; undecylenic acid and salts thereof; hexetidine; 5-bromo-5-nitro-1,3-dioxane; 2- bromo-2-nitropropane-1 ,3,-diol; dichlorobenzyl alcohol; triclocarban; p-chloro-m-cresol; triclosan; chloroxylenol; imidazolidinyl urea; polyaminopropyl biguanide; phenoxyethanol, methenamine; quaternium-15; climbazole; DMDM hydantoin; benzyl alcohol; piroctone olamine; bromochlorophene; o- cymen-5-ol; methylchloroisothiazolinone; methylisothiazolinone; chlorophene; chloroacetamide; chlorhexidine; chlorhexidine diacetate; chlorhexidine digluconate; chlorhexidine dihydrochloride; phenoxyisopropanol; alkyl (C12-C22) trimethyl ammonium bromide and chlorides; dimethyl oxazolidine; diazolidinyl urea; hexamidine; hexamidine diisethionate; glutaral; 7-ethylbicyclooxazolidine; chlorphenesin; sodium hydroxymethylglycinate; silver chloride; benzethonium chloride; benzalkonium chloride; benzalkonium bromide; benzylhemiformal; iodopropynyl butylcarbamate; ethyl lauroyl arginate HCI; citric acid and silver citrate.

[0098] Other antimicrobial agents that may be added to the compositions of the present disclosure include non-traditional antimicrobial agents that are known to exhibit antimicrobial effects in addition to their primary functions, but that have not historically been recognized as antimicrobial agents by regulatory bodies (such as on the European Union’s Annex V list). Examples of these non-traditional antimicrobial agents include, but are not limited to, hydroxyacetophenone, caprylyl glycol, sodium coco- PG dimonium chloride phosphate, phenylpropanol, lactic acid and salts thereof, caprylhydroxamic acid, levulinic acid and salts thereof, sodium lauroyl lactylate, phenethyl alcohol, sorbitan caprylate, glyceryl caprate, glyceryl caprylate, ethylhexylglycerin, p-anisic acid and salts thereof, gluconolactone, decylene glycol, 1 ,2-hexanediol, glucose oxidase and lactoperoxidase, leuconostoc / radish root ferment filtrate and glyceryl laurate.

[0099] The amount of the antimicrobial agents in the compositions is dependent on the relative amounts of other components present within the composition. For example, in some embodiments, an antimicrobial agent can be present in the compositions in an amount between about 0.001 % to about 5% (by total weight of the composition), in some embodiments between about 0.01 to about 3% (by total weight of the composition), and in some embodiments, between about 0.05% to about 1 .0% (by total weight of the composition). In some embodiments, the antimicrobial agent can be present in the composition in an amount less than 0.2% (by total weight of the composition). However, in some embodiments, the composition can be substantially free of any antimicrobial agents. Thus, in some embodiments, the composition does not include a traditional antimicrobial agent or a non-traditional antimicrobial agent.

[0100] Other suitable additives that may be included in the compositions of the present disclosure include compatible colorants, deodorants, emulsifiers, anti-foaming agents (when foam is not desired), lubricants, skin conditioning agents, skin protectants and skin benefit agents (e.g., aloe vera and tocopheryl acetate), solvents (e.g., water soluble glycol and glycol ethers, glycerin, water soluble polyethylene glycols, water soluble polyethylene glycol ethers, water soluble polypropylene glycols, water soluble polypropylene glycol ethers, dimethylisosorbide), solubilizing agents, suspending agents, builders, (e.g., alkali and alkaline earth metal salts of carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate hydrogen sulfate), wetting agents, chelators, propellants, dyes and / or pigments, and combinations thereof.

[0101] Compositions can alternatively or additionally be administered to a user through a delivery mechanism such as, for example, a wipe substrate or by being applied to at least a portion of an absorbent article that can deliver the composition to the user. For example, in one embodiment, the composition can be applied to a top sheet of a feminine care pad. In some embodiments, the composition could be administered to a user through a suppository.

[0102] In certain embodiments, suitable substrates, and applicators for delivery to a user include a web, such as a wet laid tissue web or air laid web, gauze, cotton swab, transdermal patch, container or holder. Particularly preferred applicators include fibrous webs, including flushable and non-flushable cellulosic webs and nonwoven webs of synthetic fibrous material. Useful webs may be wet laid, air laid, meltblown, or spunbonded. Suitable synthetic fibrous material includes meltblown polyethylene, polypropylene, copolymers of polyethylene and polypropylene, bicomponent fibers including polyethylene or polypropylene, and the like. Useful nonwoven webs may be meltblown, coform, spunbond, airlaid, hydroentangled nonwovens, spunlace, bonded carded webs.

[0103] In certain embodiments, particularly those in which the composition is applied to a web, it may be desirable that the formulation provide certain physical attributes, such as having a smooth, lubricious, non-greasy feel; the ability to at least partially transfer from the web to the user; the capability to be retained on the web at about room temperature; or the ability to be compatible with the web manufacturing process. In certain embodiments, it is preferred that at least a portion of the composition is transferred from the tissue to the user for the best therapeutic effect. The composition may be applied to a web during formation of the web or after the web has been formed and dried, often referred to as off-line or post-treatment. Suitable methods of applying the composition to a web include methods known in the art such as gravure printing, flexographic printing, spraying, WEKO™, slot die coating, or electrostatic spraying. One particularly preferred method of offline application is rotogravure printing.

[0104] The present invention may be better understood by reference to the following examples.

[0105] EXAMPLES

[0106] EXAMPLE 1 : PREPARATION OF THE LACTOBACILLUS SPP. BACTERIA, DELETERIOUS BACTERIA, AND THE POSTBIOTIC AND POSTBIOTIC / PROBIOTIC GELS

[0107] Freezer stocks of Lactobacillus crispatus KC18-1174-1 , Lactobacillus gasseri KC18-1132-2, Lactobacilus iners KC17-4296-7, Enterococcus faecalis KC17-4344-5, and Staphylococcus aureus KC17-4367-2 were utilized throughout the following examples.

[0108] Fig. 1 demonstrates the production of both the postbiotic and postbiotic / probiotic containing gels. The three Lactobacillus strains were added to a 5.0 mL glass rectangular growth chamber containing De Man-Rogosa-Sharpe (MRS) culture media with glucose at a concentration of 1 x 106CFU / mL each. The chamber including MRS culture media with glucose was incubated at 37 °C anaerobically overnight until stationary. All culturing was performed anaerobically to mimic a normal vaginal-like environment. The bacteria were pelleted by centrifugation (5,000 x g) for 10 minutes. The pellet was resuspended and washed in 2 x PBS. The bacteria were then subcultured on MRS culture media with glycogen but no glucose. The subculture was then incubated at 37 °C anaerobically overnight until stationary. The bacteria were then again pelleted by centrifugation (5,000 x g) for 10 minutes, resuspended, and washed in 2 x PBS. The bacteria were then subcultured on Minimal Lactobacillus Media (MLM) broth with glycogen but no glucose and incubated overnight at 37°C anaerobically.

[0109] The Minimal Lactobacillus Media (MLM) culture media was prepared by combining 25 mM MOPS (pH 6.8), M9 minimal salts (1X), Sigma V1 Vitamin Kit, and Amino Acids Mix Solution (1X). It is further supplemented with 1mM MgCI2-6H20, 1mM MnCl2-4H20, 1 mM FeSO4- 7H2O, 250 pM tryptophan, 250 pM cysteine, 250 pM CaCl2, 250 pM ZnSO4-7H2O, 50 pM CUSO4-5H20, 1 mM TWEEN 80, and 125-mM bovine glycogen. Guanine, thymine, cytidine, 2'-deoxyadenosine, and 2'- deoxyuridine were added to a final concentration of 0.5 mM each. The Minimal Lactobacillus Media (MLM) culture media was filter sterilized through a 2 micron vacuum filter prior to use. The bacteria were then pelleted, resuspended, and washed in 2 x PBS. This process was then repeated, except that after the pellet was formed, the supernatant was decanted from the pellet and recentrifuged at (50,000 x g) for 30 minutes. The supernatant was then passed over a Sephadex G-10 column (10 cm x 3 cc2). One column volume was collected, and this constituted the final postbiotic mixture. The gel exclusion column removed any remaining high molecular weight materials and unmetabolized glycogen.

[0110] The final postbiotic supernatant was then then added to two aliquots of 0.7 % agarose. The first aliquot contained only the 0.7% agarose and the postbiotic supernatant and the second aliquot contained the 0.7% agarose, the final postbiotic supernatant, and a 1 x 104CFU / mL sample of probiotic including an equal mixture of Lactobacillus crispatus KC18-1174-1 , Lactobacillus gasseri KC18-1132-2, and Lactobacilus iners KC17-4296-7. Thus, the second aliquot included not only the postbiotic supernatant, but a probiotic including equal concentrations of the three Lactobacillus spp. used to make the postbiotic.

[0111] Solidifying the postbiotic extract or the postbiotic / probiotic mixture in 0.7% agarose ensured that small organic modules were freely diffusible into the bulk media, but that the bacteria would be entrapped. The 0.7% agarose gels were prepared by adding 45 °C molten 1 .4% agarose to an equal volume of postbiotic extract and allowing the mixture to solidify in the bottom of a cuvette.

[0112] EXAMPLE 2: GROWTH EXPERIMENTS

[0113] After formation of the postbiotic and postbiotic / probiotic gels as described in Example 1 , all samples were subjected to growth experiments immediately. First, samples were prepared to determine the effect of growth of L. crispatus in Minimal Lactobacillus Media (MLM) broth. Samples were prepared according to Table 1 :

[0114] Table 1 : Samples for L crispatus growth experiments.

[0115] The results of the L. crispatus growth experiments are shown in Fig. 2 and are quantified below in Table 2.

[0116] Table 2: Growth variables derived from Fig. 2.

[0117] As demonstrated by Fig. 2 and Table 2, the presence of either the postbiotic extract gel or the postbiotic / probiotic extract gel improved the growth kinetics of L crispatus, as evidenced by a reduction in lag time, a faster doubling rate during the exponential growth phase, and a higher stationary phase bacterial concentration when compared to growth of L. crispatus alone or when the pH of the media was lowered to match the pH of the extract.

[0118] Next, samples prepared according to Table 3 were tested to determine the effect of growth of E. faecalis in Minimal Lactobacillus Media (MLM) broth. Samples were prepared according to Table 3. Table 3: Samples for E. faecalis growth experiments.

[0119] The results of the E. faecalis growth experiments are shown in Fig. 3 and are quantified below in Table 4.

[0120] Table 4: Growth variables derived from Fig. 3.

[0121] As demonstrated by Fig. 3 and Table 4, the presence of either the postbiotic extract gel or the postbiotic / probiotic extract gel shows a marked delay in the onset of growth and a significant reduction in the final E. faecalis concentration in the stationary phase. Growth kinetics in the presence of the postbiotic gel were similar to the growth at a reduced pH, but growth was significantly reduced beyond those levels in the presence of the postbiotic / prebiotic extract gel.

[0122] A control experiment was performed to ensure that the Lactobacilli added to the agarose gel were not moving from the gel to the bulk media and adding to the increase in optical density during the growth experiments. Fig. 4 indicates that over the course of the growth experiments, lactobacilli remain entrapped in the gel. Hence all changes in optical density in Figs. 2-3 are due to growth of bacterial spp. added to the bulk medium and not to diffusional growth of the lactobacilli added to the extract gel.

[0123] EXAMPLE 3: INTERACTION OF LACTOBACILLUS SPP.

[0124] Samples of various Lactobacillus spp. and mixtures thereof were prepared to determine the effect of growth of L. crispatus in Minimal Lactobacillus Media (MLM) broth. Samples were prepared according to Table 5:

[0125] Table 5: Samples of various Lactobacillus spp. and mixtures thereof forL. Crispatus growth experiments

[0126] The results of the various Lactobacillus spp. samples and their effect on L crispatus growth are shown in Fig. 5 and are quantified below in Table 6

[0127] Table 6: Growth variables derived from Fig. 5.

[0128]

[0129] As demonstrated by Fig. 5 and Table 6, the positive growth effect on the addition of the probiotic to the postbiotic extract is due to the interaction between the three Lactobacillus spp. (i.e, L. iners, L gasseri, and L. crispatus), which may function as a “mini-microbiome.” Thus, as demonstrated by Fig. 5 and Table 6, a probiotic that is composed of a single Lactobacillus spp. is less effective in stimulating growth of L crispatus than a probiotic compound composed of a mixture of L. iners, L. crispatus, and L. gasseri. This may indicate that the interaction between the three spp. also produces a different postbiotic mixture, either in the concentration of components or in the presence or absence of unique components.

[0130] EXAMPLE 4: GLYCOGEN PASSAGING

[0131] Samples of Lcrispatus in the presence of the postbiotic / probiotic formulation were prepared with or without passage on glycogen as the sole carbon source to determine the effect of growth of L. crispatus in Minimal Lactobacillus Media (MLM) broth. Samples were prepared according to Table 7:

[0132] T able 7 : Samples of L. crispatus in the presence of the postbiotic / probiotic formulation prepared with or without passage on glycogen. The results of the various samples of Table 7 tested for L crispatus growth are shown in Fig. 6 and demonstrated below in Table 8.

[0133] Table 8: Growth variables derived from Fig. 6.

[0134] Likewise, samples prepared according to Table 7 were tested to determine the effect of growth of E. faecalis in Minimal Lactobacillus Media (MLM) broth. The results of the various samples of Table 7 tested for E. faecalis growth are shown in Fig. 7 and demonstrated below in Table 9.

[0135] Table 9: Growth variables derived from Fig. 7.

[0136] As demonstrated by Figs. 6-7 and Tables 8-9, passaging the Lactobacillus vaginal minimicrobiome bacteria using glycogen as a sole carbon source is required to see the maximal effect of the postbiotic / probiotic mixture on the growth of L. crispatus and the growth inhibition of E. faecalis. As seen in Fig. 6, when glycogen is utilized as part of the formulation production, there is a marked increase in final amount of L. crispatus growth, the rate of exponential growth, and a measurable decrease in the growth lag time. The postbiotic / probiotic mixture produced with glucose only, and thus with no glycogen, does show a comparable reduction in lag time compared to the glycogen formulation, but the final growth rate and bacterial concentration are comparable to the control.

[0137] As seen in Fig. 7, growth of E. faecalis shows that there are significant differences between the glycogen -prepared postbiotic / probiotic formulation and the formulation prepared without the passage of the bacteria on glycogen. Relative to the growth control curve, growth of the bacterium in the (+) glycogen formulation shows a marked decrease in final bacterial growth concentration and an increase in lag phase, whereas the (-) glycogen postbiotic / probiotic formulation is significantly less effective in both measures.

[0138] EXAMPLE EMBODIMENTS

[0139] Example Embodiment 1 : A method for producing a microbiome modulating composition, the method comprising: preparing a culture from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof; passaging the culture on a glycogen carbon source to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof; and adding the postbiotic ferment to a carrier.

[0140] Example Embodiment 2: The method of example embodiment 1 , wherein a probiotic comprising a mixture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof is further added to the microbiome modulating composition.

[0141] Example Embodiment 3: The method as in any preceding embodiment, wherein a prebiotic is added to the microbiome modulating composition.

[0142] Example Embodiment 4: The method as in any preceding embodiment, wherein the prebiotic comprises at least one Lactobacillus spp. bacteria-specific prebiotic, at least one Limosilactobacillus spp. bacteria- specific prebiotic, or combinations thereof.

[0143] Example Embodiment 5: The method as in any preceding embodiment, wherein the microbiome modulating composition has a pH of from about 3.8 to about 5.0. Example Embodiment 6: The method as in any preceding embodiment, wherein the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof include a mixture of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillus johnsonii, Limosilactobacillus vaginalis, Limosilactobacillus reuteri, Limosilactobacillus fermentum, or Limosilactobacillus rhamnosus.

[0144] Example Embodiment 7: The method as in any preceding embodiment, wherein the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof include a mixture of Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus iners.

[0145] Example Embodiment 8: The method as in any preceding embodiment, wherein the microbiome modulating composition is a vaginal insertable gel.

[0146] Example Embodiment 9: The method as in any preceding embodiment, wherein the cultures from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof are initially prepared on De Man-Rogosa-Sharpe (MRS) culture media with glucose.

[0147] Example Embodiment 10: The method as in any preceding embodiment, wherein the glycogen carbon source is a Minimal Lactobacillus Media (MLM) culture media without glucose.

[0148] Example Embodiment 1 1 : The method as in any preceding embodiment, further comprising pelleting the culture to form a pellet, washing the pellet with 2 x PBS solution, and re-passaging the culture on a glycogen carbon source.

[0149] Example Embodiment 12: The method as in any preceding embodiment, wherein the culture was passaged on the glycogen carbon source under hypogravity conditions.

[0150] Example Embodiment 13: A microbiome modulating composition comprising: a carrier; and a postbiotic ferment produced from passaging a culture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof on a glycogen carbon source.

[0151] Example Embodiment 14: The microbiome modulating composition of example embodiment 13, wherein the carrier is an aqueous solution. Example Embodiment 15: The microbiome modulating composition as in any preceding embodiment, wherein the microbiome modulating composition is a gel, a liquid, a cream, a spray, or a suppository.

[0152] Example Embodiment 16: The microbiome modulating composition as in any preceding embodiment, wherein the microbiome modulating composition is a vaginal insertable gel.

[0153] Example Embodiment 17: The microbiome modulating composition as in any preceding embodiment, wherein the postbiotic ferment comprises about 0.1 % wt / vol to about 10% wt / vol of the microbiome modulating composition by volume.

[0154] Example Embodiment 18: The microbiome modulating composition as in any preceding embodiment, wherein the carrier comprises greater than about 90% wt / vol of the microbiome modulating composition by volume.

[0155] Example Embodiment 19: The microbiome modulating composition as in any preceding embodiment, wherein the microbiome modulating composition further comprises a probiotic comprising a mixture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof.

[0156] Example Embodiment 20: The microbiome modulating composition as in any preceding embodiment, wherein the microbiome modulating composition further comprises a prebiotic.

[0157] Example Embodiment 21 : The microbiome modulating composition as in any preceding embodiment, wherein the prebiotic comprises at least one Lactobacillus spp. bacteria-specific prebiotic, at least one Limosilactobacillus spp. bacteria-specific prebiotic, or combinations thereof.

[0158] Example Embodiment 22: The microbiome modulating composition as in any preceding embodiment, wherein the microbiome modulating composition further comprises at least one of an additional urogenital prebiotic, a surfactant, an ester, a humectant, a pH adjuster, a rheology modifier, a gelling agent, a nitrogen source, and an antimicrobial agent.

[0159] Example Embodiment 23: The microbiome modulating composition as in any preceding embodiment, wherein when the microbiome modulating composition is applied to a culture of L. crispatus on Minimal Lactobacillus Media (MLM) culture media, the culture of L crispatus demonstrates a lag time in growth of about 105 minutes or less.

[0160] Example Embodiment 24: The microbiome modulating composition as in any preceding embodiment, wherein when the microbiome modulating composition is applied to a culture of L. crispatus on Minimal Lactobacillus Media (MLM) culture media, the culture of L. crispatus demonstrates a growth rate during exponential growth of about 0.004 AOD / min or greater.

[0161] Example Embodiment 25: The microbiome modulating composition as in any preceding embodiment, wherein when the microbiome modulating composition is applied to a culture of Lcrispatus on Minimal Lactobacillus Media (MLM) culture media, a final bacterial concentration (OD at 600 nm) of L. crispatus is about 1 .55 or greater.

[0162] Example Embodiment 26: The microbiome modulating composition as in any preceding embodiment, wherein when the microbiome modulating composition is applied to a culture of E. faecalis on Minimal Lactobacillus Media (MLM) culture media, the culture of E. faecalis demonstrates a growth rate during exponential growth of about 0.0006 AOD / min or less.

[0163] Example Embodiment 27: The microbiome modulating composition as in any preceding embodiment, wherein when the microbiome modulating composition is applied to a culture of E. faecalis on MLM culture media, the final bacterial concentration (OD at 600 nm) of E. faecalis is about 0.3 or less.

[0164] Example Embodiment 28: A method for treating microbial dysbiosis of an urogenital area or a skin area of a user, the method comprising: applying a microbiome modulating composition to an urogenital or a skin area of a user, the composition comprising: a carrier; and a postbiotic ferment produced from passaging a culture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof on a glycogen carbon source.

[0165] Example Embodiment 29: The method of example embodiment 28, further comprising applying the microbiome modulating composition to a substrate, the substrate comprising a wipe or at least a portion of an absorbent article.

[0166] Example Embodiment 30: The method as in any preceding embodiment, further comprising promoting a healthy microflora balance of the urogenital area or the skin area of the user, wherein promoting the healthy microflora balance of the urogenital area or the skin area of the user comprises promoting a growth of Lactobacillus spp. bacteria relative to a growth of one or more types of Enterococcus spp. bacteria, Staphylococcus spp. bacteria, Escherichia spp. bacteria, Gardnerella spp. bacteria, Streptococcus spp. bacteria, or mixtures thereof.

[0167] Example Embodiment 31 : The method as in any preceding embodiment, wherein a probiotic comprising a mixture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof is further added to the microbiome modulating composition

Claims

WHAT IS CLAIMED IS:1 . A method for producing a microbiome modulating composition, the method comprising: preparing a culture from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof; passaging the culture on a glycogen carbon source to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof; and adding the postbiotic ferment to a carrier.

2. The method of claim 1 , wherein a probiotic comprising a mixture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof is further added to the microbiome modulating composition.

3. The method of claim 1 , wherein a prebiotic is added to the microbiome modulating composition.

4. The method of claim 3, wherein the prebiotic comprises at least one Lactobacillus spp. bacteria- specific prebiotic, at least one Limosilactobacillus spp. bacteria-specific prebiotic, or combinations thereof.

5. The method of claim 1 , wherein the microbiome modulating composition has a pH of from about 3.8 to about 5.0.

6. The method of claim 1 , wherein the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof include a mixture of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillus johnsonii, Limosilactobacillus vaginalis, Limosilactobacillus reuteri, Limosilactobacillus fermentum, or Limosilactobacillus rhamnosus.

7. The method of claim 1 , wherein the at least two Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof include a mixture of Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus iners8. The method of claim 1 , wherein the microbiome modulating composition is a vaginal insertable gel.

9. The method of claim 1 , wherein the cultures from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof are initially prepared on De Man- Rogosa-Sharpe (MRS) culture media with glucose.

10. The method of claim 1 , wherein the glycogen carbon source is a Minimal Lactobacillus Media (MLM) culture media without glucose.

11. The method of claim 1 , further comprising pelleting the culture to form a pellet, washing the pellet with 2 x PBS solution, and re-passaging the culture on a glycogen carbon source.

12. The method of claim 1 , wherein the culture was passaged on the glycogen carbon source under hypogravity conditions.

13. A microbiome modulating composition comprising: a carrier; and a postbiotic ferment produced from passaging a culture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof on a glycogen carbon source.

14. The microbiome modulating composition of claim 13, wherein the carrier is an aqueous solution.

15. The microbiome modulating composition of claim 13, wherein the microbiome modulating composition is a gel, a liquid, a cream, a spray, or a suppository.

16. The microbiome modulating composition of claim 13, wherein the microbiome modulating composition is a vaginal insertable gel.

17. The microbiome modulating composition of claim 13, wherein the postbiotic ferment comprises about 0.1 % wt / vol to about 10% wt / vol of the microbiome modulating composition.

18. The microbiome modulating composition of claim 13, wherein the carrier comprises greater than about 90% wt / vol of the microbiome modulating composition.

19. The microbiome modulating composition of claim 13, wherein the microbiome modulating composition further comprises a probiotic comprising a mixture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof.

20. The microbiome modulating composition of claim 13, wherein the microbiome modulating composition further comprises a prebiotic.

21. The microbiome modulating composition of claim 20, wherein the prebiotic comprises at least one Lactobacillus spp. bacteria-specific prebiotic, at least one Limosilactobacillus spp. bacteria- specific prebiotic, or combinations thereof.

22. The microbiome modulating composition of claim 13, wherein the microbiome modulating composition further comprises at least one of an additional urogenital prebiotic, a surfactant, an ester, a humectant, a pH adjuster, a rheology modifier, a gelling agent, a nitrogen source, and an antimicrobial agent.

23. The microbiome modulating composition of claim 13, wherein when the microbiome modulating composition is applied to a culture of L.crispatus on Minimal Lactobacillus Media (MLM) culture media, the culture of L crispatus demonstrates a lag time in growth of about 105 minutes or less.

24. The microbiome modulating composition of claim 13, wherein when the microbiome modulating composition is applied to a culture of L.crispatus on Minimal Lactobacillus Media (MLM) culture media, the culture of L. crispatus demonstrates a growth rate during exponential growth of about 0.004 AOD / min or greater.

25. The microbiome modulating composition of claim 13, wherein when the microbiome modulating composition is applied to a culture of L crispatus on Minimal Lactobacillus Media (MLM) culture media, a final bacterial concentration (OD at 600 nm) of L. crispatus is about 1 .55 or greater.

26. The microbiome modulating composition of claim 13, wherein when the microbiome modulating composition is applied to a culture of E. faecalis on Minimal Lactobacillus Media (MLM) culture media, the culture of E. faecalis demonstrates a growth rate during exponential growth of about 0.0006 AOD / min or less.

27. The microbiome modulating composition of claim 13, wherein when the microbiome modulating composition is applied to a culture of E. faecalis on Minimal Lactobacillus Media (MLM) culture media, the final bacterial concentration (OD at 600 nm) of E.faecalis is about 0.3 or less.

28. A method for treating microbial dysbiosis of an urogenital area or a skin area of a user, the method comprising: applying a microbiome modulating composition to an urogenital or a skin area of a user, the composition comprising: a carrier; and a postbiotic ferment produced from passaging a culture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof on a glycogen carbon source.

29. The method of claim 29, further comprising applying the microbiome modulating composition to a substrate, the substrate comprising a wipe or at least a portion of an absorbent article.

30. The method of claim 29, further comprising promoting a healthy microflora balance of the urogenital area or the skin area of the user, wherein promoting the healthy microflora balance of the urogenital area or the skin area of the user comprises promoting a growth of Lactobacillus spp. bacteria relative to a growth of one or more types of Enterococcus spp. bacteria,Staphylococcus spp. bacteria, Escherichia spp. bacteria, Gardnerella spp. bacteria, Streptococcus spp. bacteria, or mixtures thereof.

31. The method of claim 29, wherein a probiotic comprising a mixture of at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof is further added to the microbiome modulating composition.

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