Hypogravity preparation of postbiotic ferments

Abstract

A method for producing a postbiotic ferment and a microbiome modulating composition including the postbiotic ferment are disclosed herein. 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 carbon source in a reduced gravity environment to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof. The reduced gravity environment maintains a gravitational load of an average gravity value of about 0.0 x g to about 0.25 x g. 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 including the postbiotic ferment prepared in a reduced gravity environment 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] HYPOGRAVITY PREPARATION OF POSTBIOTIC FERMENTS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to the benefit of U.S. Provisional Application No. 63 / 718,009, 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 in 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] The use of microbiome modulating compounds such as probiotics, prebiotics, and postbiotic ferments are known in the art and have been an alternative means for maintenance of urogenital health and treatment of common vaginal infections. However, the production of microbiome modulating compounds, such as probiotics, prebiotics, and postbiotic ferments can often be tedious and result in compounds that lack high levels of efficacy, such as the efficacy demonstrated by antibiotics.

[0009] Accordingly, a need exists for methods for producing microbiome modulating compounds, such as probiotics, prebiotics, and postbiotic ferments, that have improved efficacy.

[0010] SUMMARY OF THE DISCLOSURE

[0011] In general, the present disclosure is directed towards a method for producing a postbiotic ferment and compositions thereof. 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 carbon source in a reduced gravity environment to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof. The reduced gravity environment maintains a gravitational load of an average gravity value of about 0.0 x g to about 0.25 x g. In certain example embodiments, the average gravity value is from about 0.0 x g to about 0.005 x g, such as from about 0.0012 x g to about 0.0042 x g.

[0012] In certain example embodiments, the carbon source is a glycogen carbon source. In some example embodiments, the glycogen carbon source is a Minimal Lactobacillus Media (MLM) culture media without glucose.

[0013] 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.

[0014] It should be understood by one of ordinary skill in the art that the method of the present disclosure may comprise one or more optional steps that may be manipulated to form varying characteristics of the postbiotic ferment. For instance, in certain example embodiments, 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. In other 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 carbon source in the reduced gravity environment. In some example embodiments, the method further comprises pelleting the culture to form a pellet and harvesting the supernatant from the pellet to form a final postbiotic ferment.

[0015] The method of the present disclosure may provide a postbiotic ferment that has unexpected efficacy in enhancing the growth of beneficial spp., such as Lactobacillus spp. bacteria, while inhibiting the growth of deleterious, pathogenic species. For instance, in certain example embodiments, when the postbiotic ferment is applied to a beneficial species, the beneficial species demonstrates an exponential growth lag time of about 75 minutes to about 100 minutes less than an exponential growth lag time of a beneficial species that had no postbiotic ferment applied. Likewise, in other example embodiments, when the postbiotic ferment is applied to a beneficial species, the beneficial species demonstrates an exponential growth lag time of about 20 minutes to about 50 minutes less than an exponential growth lag time of a beneficial species that had a postbiotic ferment produced in a normal gravity environment applied. In other example embodiments, when the postbiotic ferment is applied to a deleterious species, the deleterious species demonstrates an exponential growth lag time of about 150 minutes to about 750 minutes more than an exponential growth lag time of a deleterious species that had no postbiotic ferment applied. In certain example embodiments, when the postbiotic ferment is applied to a deleterious species, the deleterious species demonstrates an exponential growth lag time of about 75 minutes to about 650 minutes more than an exponential growth lag time of a deleterious species that had a postbiotic ferment produced in a normal gravity environment applied.

[0016] Likewise, the method of the present disclosure may also enhance the final concentration or bacterial density of a beneficial species while decreasing the final concentration or bacterial density of a deleterious, pathogenic species. For instance, in certain example embodiments, when the postbiotic ferment is applied to a beneficial species, a ratio of a final bacterial density (OD at 600 nm) of the beneficial species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a beneficial species without a postbiotic ferment is from about 1 .6 to about 2.0. In other example embodiments, when the postbiotic ferment is applied to a beneficial species, a ratio of a final bacterial density (OD at 600 nm) of the beneficial species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a beneficial species with a postbiotic ferment produced in a normal gravity environment is from about 1.1 to about 1 .55. In certain example embodiments, when the postbiotic ferment is applied to a deleterious species, a ratio of a final bacterial density (OD at 600 nm) of the deleterious species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a deleterious species without a postbiotic ferment is from about is from about 0.01 to about 0.3. In some example embodiments, when the postbiotic ferment is applied to a deleterious species, a ratio of a final bacterial density (OD at 600 nm) of the deleterious species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a deleterious species with a postbiotic ferment produced in a normal gravity environment is from about 0.04 to about 0.5.

[0017] The present disclosure is also generally directed to 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 carbon source in a reduced gravity environment. In certain example embodiments, the carrier is an aqueous solution. In other example embodiments, the microbiome modulating composition is a gel, a liquid, a cream, a spray, or a suppository. In certain suitable example embodiments, the microbiome modulating composition is a vaginal insertable gel.

[0018] In some 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.

[0019] 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 some example embodiments, the microbiome modulating composition further comprises a prebiotic, such as at least one Lactobacillus spp. bacteria-specific prebiotic, at least one Limosilactobacillus spp. bacteria-specific prebiotic, or combinations thereof.

[0020] In other example embodiments, the microbiome modulating composition has a pH of from about 3.8 to about 5.0. 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.

[0021] The present disclosure is also generally directed to 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. In other 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 some example embodiments, the method further comprises reducing the severity of an infection or a microbial dysbiosis of the urogenital area or the skin area of a user, reducing a reoccurrence frequency of an infection or a microbial dysbiosis of the urogenital area or the skin area of a user, and / or returning the urogenital or the skin area of the user to a microbial homeostasis. In certain example embodiments, the microbial dysbiosis of the urogenital or the skin area of a user includes bacterial vaginosis, candidal vaginitis ("yeast"), trichomonas vaginitis, urinary tract infections, bladder infections, or a combination thereof. In other example embodiments, the microbial dysbiosis of the urogenital or the skin area of a user includes diaper rash, diaper dermatitis, inflammation, redness, or a combination thereof. In some example embodiments, the microbial dysbiosis of the urogenital or the skin area of a user includes nasal skin irritation.

[0022] 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.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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:

[0025] Figure 1 graphically illustrates the average value of the gravity vector < g > versus time for the first 2 hours of the RPM run to produce postbiotic ferments of Lactobacillus spp. bacteria as described in Example 1 .

[0026] Figure 2 graphically illustrates the coverage of x, y, z space as sampled during the RPM run as described in Example 1 .

[0027] Figure 3 graphically illustrates the production of the postbiotic ferments utilizing hypogravity conditions as described in Example 2.

[0028] Figure 4 graphically illustrates the production of the postbiotic ferments utilizing normal gravity conditions as described in Example 3.

[0029] Figure 5 graphically illustrates the growth of Lactobacillus spp. bacteria (including Lactobacillus crispatus “Lc”, Lactobacillus iners “Li”, and Lactobacillus gaseri “Lg”) in the A) presence of the hypogravity postbiotic, B) presence of the normal gravity postbiotic, and C) in the absence of any added postbiotic as described in Example 3. Figure 6 graphically illustrates the growth of deleterious vaginal flora (including Enterococcus faecalis “Ef”, Staphylococcus aureus “Sa", and Candida albicans “Ca”) in the A) absence of any postbiotic, B) presence of the normal gravity postbiotic, and C) presence of the hypogravity postbiotic as described in Example 3.

[0030] Figure 7 graphically illustrates the average value of the gravity vector < g > versus time for the five varied hypogravity postbiotics produced as described in Example 4.

[0031] Figure 8 graphically illustrates the growth of L crispatus in the presence of hypogravity postbiotic formulations produced under average stimulated hypogravity vector < g > values between 0 x g and 1 .0 x g as described in Example 4.

[0032] Figure 9 graphically illustrates the growth of S. aureus in the presence of hypogravity postbiotic formulations produced under average simulated hypogravity vector < g > values between 0 x g and 1 .0 x g as described in Example 4.

[0033] Figure 10 graphically illustrates the growth of S. epidermidis in the presence of a hypogravity postbiotic, a normal gravity postbiotic, and in the absence of a postbiotic as described in Example 5.

[0034] Figure 11 graphically illustrates the growth of R. gnavus in the presence of a hypogravity postbiotic, a normal gravity postbiotic, and in the absence of a postbiotic as described in Example 5.

[0035] 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.

[0036] DEFINITIONS

[0037] When introducing elements of the present disclosure or the preferred embodiment(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. As used herein, the terms “includes” and “including” are intended to be inclusive in a manner similar to the term “comprising.” Similarly, the term “or” is generally intended to be inclusive (i.e., “A or B” is intended to mean “A or B or both”). Approximating language, as used herein throughout the specification and claims, is applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about,” “approximately,” and “substantially,” are not to be limited to the precise value specified. In at least some instances, the approximating language may correspond to the precision of an instrument for measuring the value. For example, the approximating language may refer to being within a ten percent (10%) margin. As used herein, the term “comprising” or “including” or “having” are inclusive or open-ended and do not exclude additional unrecited elements, compositional components, or method steps. Accordingly, the terms “comprising" or “including" or “having” encompass the more restrictive terms “consisting essentially of' and “consisting of.”

[0038] As used herein, the term “inhibit" generally means to reduce by a measurable amount or to prevent entirely.

[0039] As used herein, the term “urogenital” refers to the vaginal canal, vulva, vagina, urethra, urinary tract, bladder, perineal, anal, and surrounding areas.

[0040] As used herein, the term “skin” refers to epithelium, mucous membranes, and any other tissue forming the outer layer of a body's surface.

[0041] As used herein, the term “user” or “subject” refers to a person who is receiving the composition of the present disclosure.

[0042] 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, such as the hypogravity postbiotic ferment 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.

[0043] 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.

[0044] 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.

[0045] 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). 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.

[0046] As used herein, the term “hypograviotic” refers to any postbiotic ferment or mixture that is prepared from growing a culture of bacteria under hypogravity or reduced gravity conditions.

[0047] DETAILED DESCRIPTION OF THE DISLOSURE

[0048] It is to be understood by one of ordinary skill in the art that the present disclosure is a description of exemplary embodiments only and is not intended as limiting the broader aspects of the present disclosure.

[0049] In general, the present disclosure is directed towards a method for producing a postbiotic ferment in a reduced gravity environment. Postbiotic ferments may include soluble and insoluble products secreted by or made of Lactobacilllus 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, shortchain fatty acids, conjugated linoleic acid, exopolysacchardies, bacteriocins, lanthipeptides, an D / L-lactic acid. The method of the present disclosure includes preparing a culture from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof and passaging the culture on a carbon source in a reduced gravity environment to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof. Notably, without intending to be limited by theory, the present inventors have discovered that the use of reduced gravity (i.e. “hypogravity”) environments, especially in the low milligravity to microgravity regime, may alter the physiology of the resulting postbiotic ferments (i.e. “hypogravity postbiotics” or “hypograviotics”).

[0050] Hypogravity, whether actual or simulated, is a powerful technique used to study biophysical and biochemical phenomena. Hypogravity-based research has influenced many diverse biological fields including protein crystallization, drug development and pharmacodynamics, plant growth physiology, proteomics, host-pathogen interactions and virulence, tissue engineering, cell biology, mammalian development, human cellular physiology / gene expression, and cancer. Historically, however, the use of hypogravity-based research has been specifically helpful for the fields of space exploration. For instance, hypogravity-based research has been utilized to determine the various roles and effects of microgravity on human and plant life, especially in view of potential future space exploration advancements.

[0051] Although traditional hypogravity studies have previously been limited to space fight or expensive and hard to manipulate terrestrial systems, the development of random positioning machines (i.e., “RPM”) and other clinostats have allowed hypogravity to emerge as a useful tool in the formation of 3-D cultures without significant infrastructure cost.

[0052] Thus, the method of the present disclosure, in certain embodiments, may utilize a random positioning machine or a clinostat to create a reduced gravity environment for the production of postbiotic ferments (i.e. “hypogravity postbiotics” or “hypograviotics”). A random positioning machine generally includes two independently rotating frames with an inner axis rotational speed and an outer axis rotational speed. The random positioning machine may collect x, y, z positional / orientation data and the value of the three accelerometer vectors all as a function of time. The final average value of the reduced gravity vector may be calculated using the individual vectors obtained from an accelerometer according to:

[0053] One of ordinary skill in the art should understand that the random positioning machine can be manipulated to achieve various results. For instance, rotational speeds greater than ~4 rpm may introduce centrifugal forces which undermine the low gravity regime. Further, manipulating both the inner axis rotational speed and the outer axis rotational speed of the random positioning device may also produce different effects in the resulting hypogravity conditions. For instance, as the axial speeds decrease, the amount of time to reach a stable < g> may also decrease. Thus, because of the control afforded by the wide range of axial rotation speeds (and their ratios), it may be possible to manipulate the formation of the postbiotic ferment by controlling the hypogravity level.

[0054] The reduced gravity environment required by the present disclosure maintains a gravitational load of an average gravity value < g > of about 0.0 x g to about 0.25 x g. For instance, the average gravity value may be about 0.0 x g or greater, such as about 0.0005 x g or greater, such as about 0.00075 x g or greater, such as about 0.001 x g or greater, such as about 0.0012 x g or greater, such as about 0.0015 x g or greater, such as about 0.002 x g or greater, such as about 0.0025 x g or greater, such as about 0.003 x g or greater, such as about 0.004 x g or greater, such as about 0.0042 x g or greater, such as about 0.005 x g or greater, such as about 0.006 x g or greater, such as about 0.007 x g or greater, such as about 0.008 x g or greater, such as about 0.009 x g or greater, such as about 0.01 x g or greater, and generally less than about 0.25 x g or less, such as about 0.2 x g or less, such as about 0.15 x g or less, such as about 0.1 x g or less, such as about 0.01 x g or less, and such as 0.005 x g or less. It should be understood by one of ordinary skill in the art that an average gravity value of about 1 .0 x g is considered “normal” gravity, whereas anything under 1 .0 x g is considered “hypogravity.”

[0055] In preferred embodiments, for instance, the reduced gravity environment maintains a gravitational load of an average gravity value from about 0.0 x g to about 0.005 x g, such as from about from about 0.0012 x g to about 0.0042 x g.

[0056] Without intending to be limited by theory, the present inventors have discovered an inverse relationship between the average gravity value maintained by the reduced gravity environment and the efficacy of the resulting hypogravity postbiotic ferment (i.e., “hypograviotic”). Thus, more significant growth of beneficial species bacteria was demonstrated and more significant inhibition of growth of deleterious species was likewise demonstrated when the growth experiments occurred with a hypogravity postbiotic (i.e., “hypograviotic”) at lower average gravity values. For instance, the presence of the hypogravity postbiotic ferment (i.e., “hypograviotic”) was able to effect the exponential growth lag time, which indicates how long it took for the bacteria to start growing in the exponential phase. In certain example embodiments, when the postbiotic ferment is applied to a beneficial species, the beneficial species demonstrates an exponential growth lag time of about 75 minutes to about 100 minutes, such as about 80 minutes to about 95 minutes, such as about 85 minutes to about 90 minutes less than an exponential growth lag time of a beneficial species that had no postbiotic ferment applied. Further, in other example embodiments, when the postbiotic ferment is applied to a beneficial species, the beneficial species demonstrates an exponential growth lag time of about 20 minutes to about 50 minutes, such as about 25 minutes to about 45 minutes, such as about 30 minutes to about 40 minutes less than an exponential growth lag time of a beneficial species that had a postbiotic ferment produced in a normal gravity environment applied.

[0057] In other example embodiments, when the postbiotic ferment is applied to a deleterious species, the deleterious species demonstrates an exponential growth lag time of about 150 minutes to about 750 minutes, such as about 200 minutes to about 700 minutes, such as about 350 minutes to about 675 minutes more than an exponential growth lag time of a deleterious species that had no postbiotic ferment applied. In other example embodiments, when the postbiotic ferment is applied to a deleterious species, the deleterious species demonstrates an exponential growth lag time of about 75 minutes to about 650 minutes, such as about 90 minutes to about 600 minutes, such as about 130 minutes to about 500 minutes more than an exponential growth lag time of a deleterious species that had a postbiotic ferment produced in a normal gravity environment applied.

[0058] The final concentration or final bacterial density, measured as the optical density at 600 nm, may also be effected by the presence of the hypogravity postbiotic (i.e., “hypograviotic”) . For instance, in some example embodiments, when the postbiotic ferment is applied to a beneficial species, a ratio of a final bacterial density (OD at 600 nm) of the beneficial species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a beneficial species without a postbiotic ferment is from about 1.6 to about 2.0, such as from about 1.63 to about 1.85, such as from about 1.7 to about 1.75. In other example embodiments, when the postbiotic ferment is applied to a beneficial species, a ratio of a final bacterial density (OD at 600 nm) of the beneficial species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a beneficial species with a postbiotic ferment produced in a normal gravity environment is from about 1 .1 to about 1 .55, such as from about 1 .2 to about 1 .5, such as from about 1 .3 to about 1 .4

[0059] In certain example embodiments, when the postbiotic ferment is applied to a deleterious species, a ratio of a final bacterial density (OD at 600 nm) of the deleterious species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a deleterious species without a postbiotic ferment is from about 0.01 to about 0.3, such as from about 0.05 to about 0.25, such as from about 0. 08 to about 0.1 . In other example embodiments, when the postbiotic ferment is applied to a deleterious species, a ratio of a final bacterial density (OD at 600 nm) of the deleterious species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a deleterious species with a postbiotic ferment produced in a normal gravity environment is from about 0.04 to about 0.5, such as from about 0.1 to about 0.35, such as from about 0.25 to about 0.3.

[0060] While not intending to be limited by theory, it is believed that the superior efficacy of the hypogravity postbiotics (i.e., “hypograviotics”) in promoting the growth of beneficial species while inhibiting the growth of deleterious species is because hypogravity, especially in the low milli-g to micro- g regimes, may alter the physiology of the postbiotic ferments. For instance, certain physiological changes may include both primary and secondary metabolic changes. Such metabolic changes, in some example embodiments, may alter the composition of secondary metabolites that are produced and excreted by bacteria, such as amino acids, organic acids, and other postbiotic ferments. Notably, the excreted secondary metabolic compounds formed by Lactobacilli are detrimental to the growth of deleterious vaginal flora including, but not limited to, Staphylococcus, Escherichia, Gardnerella, Candida, and Enterococcus. Secondary metabolites can also act intracellularly to promote growth and general cellular homeostasis, thus promoting Lactobacillus growth. Hypogravity conditions can also lead to the expression of normally silent or cryptic gene clusters that give rise to molecules typically not seen under normal gravity. Thus, hypogravity postbiotics (i.e., “hypograviotics") may be more effective at treating microbiome dysbiosis, as the hypogravity formation of the postbiotic ferment may alter both the intracellular and extracellular composition of the secondary metabolite pool.

[0061] Specifically, as Lactobacillus spp. bacteria are frequently found in urogenital areas, such as the vaginal canal, or on other parts of the skin, the use of the hypogravity postbiotic ferments (i.e., “hypograviotics”) as discussed herein may provide a path to produce significantly better outcomes for not only the treatment of acute episodes of dysbiosis, but for use as a microbiome ‘maintenance’ product to lower the recurrence of dysbiotic episodes. Further, as vaginal dysbiosis differs from person to person, the method for forming the hypogravity postbiotic (i.e., “hypograviotic”) of the present invention may also have particularly suitable applications in preparing personalized postbiotic ferments created using a minimicrobiome harvested from a single individual for eventual treatment in that same individual.

[0062] The postbiotic ferments of the present invention are formed from passaging a culture of a mixture of Lactobacillus spp. bacteria or Limosilactobacillus spp. bacteria on a 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 Limosilactobacillus 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.. As discussed above, the “mini-microbiome” may be personalized such that it is tailored to a certain individual. 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. The compositions of the present disclosure generally facilitate the growth of Grampositive rod-shaped bacteria belonging to the Lactobacillus spp.. Notably, the present inventors have discovered that by utilizing a composition including a postbiotic ferment formed from a mixture of Lactobacillus spp. bacteria or Limosilactobacillus spp. bacteria passaged on a 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 often deplenishes normal bacterial flora, including the beneficial lactobacilli

[0063] 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.

[0064] The method for producing a hypogravity postbiotic ferment (i.e., “hypograviotic”) as described herein includes preparing a culture from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof and passaging the culture on a carbon source, such as a glycogen carbon source, in a reduced gravity environment to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof.

[0065] Lactobacillus spp. are used throughout the present disclosure for the production of the hypogravity postbiotic ferments (i.e., “hypograviotics”) and as the probiotic compounds that are optionally added to the microbiome modulating composition. For instance, the present inventors have found that the hypogravity postbiotic ferments (i.e., “hypograviotics”) 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 hypogravity postbiotic ferments (i.e., “hypograviotics”). 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 104to about 1010CFU / 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. 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 1 x 104to about 1 x 106CFU / mL of each Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus iners were utilized to prepare the hypogravity postbiotic ferments (i.e., “hypograviotics”).

[0066] 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 carbon source. In preferred example embodiments, the carbon source is a glycogen carbon source.

[0067] 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.

[0068] In certain preferred example 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 (1X), Sigma V1 Vitamin Kit, and Amino Acids Mix Solution (1X). It is further supplemented with 1 mM MgCh- 6H20, 1 mM MnCl2-4H20, 1 mM FeSO4- 7H2O, 250 pM tryptophan, 250 pM cysteine, 250 pM CaCI2, 250 pM ZnSO4-7H2O, 50 pM CUSO4-5H2O, 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.

[0069] In other example embodiments, the method for producing the hypogravity postbiotic ferment (i.e., “hy pog raviotic") 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 on Minimal Lactobacillus Media (MLM) media to form a pellet, washing the pellet with 2 x PBS solution, and re-passaging the culture on a carbon source in a reduced gravity environment. Thus, in some example embodiments, the culture of the at least two of any 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. In preferred example embodiments, the culture is passaged on the Minimal Lactobacillus Media (MLM) culture media (-) glucose (+) glycogen in a reduced gravity environment at least twice.

[0070] The method further comprises pelleting the culture from the Minimal Lactobacillus Media (MLM), harvesting the supernatant of the pelleted culture passaged on the 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 hypogravity postbiotic ferment (i.e. , “hypograviotic”) of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof. As referred to herein and described above, hypogravity postbiotic ferments (i.e., “hypograviotics”) 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, an 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 hypogravity postbiotic ferment (i.e., ''hypograviotic").

[0071] The present disclosure is also generally directed to a microbiome modulating composition including the hypogravity postbiotic ferment (i.e., ''hypograviotic"). The microbiome modulating composition includes 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 carbon source in a reduced gravity environment, wherein the reduced gravity environment maintains a gravitational load of an average gravity value of about 0.0 x g to about 0.25 x g.

[0072] 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 hypogravity postbiotic ferment (i.e., “hypograviotic”). 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 non-aqueous. Nonaqueous 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

[0073] 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.

[0074] Without intending to be limited by theory, the present inventors have found that microbiome modulating compositions which include only a hypogravity postbiotic ferment (i.e., “hypograviotic”) 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 or other beneficial species while inhibiting the growth of deleterious, pathogenic species. However, other therapeutic agents may be added to the composition to improve the efficacy of the hypogravity postbiotic ferments (i.e., “hypograviotics”) Suitable additional therapeutic agents often synergistically interact with the hypogravity postbiotic ferments (i.e., "hypograviotics”) 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 hypogravity postbiotic ferment (i.e., "hypograviotic”). 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 embodiments, 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 jensenll, Lactobacillus acidophilus, Lactobacillus johnsonii, Limosilactobacillus vaginalis, Limosilactobacillus reuteri, Limosilactobacillus fermentum, or Limosilactobacillus rhamnosus .

[0075] 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 hypogravity postbiotic ferment (i.e., "hypograviotic") and a probiotic, as described above, are used in combination. 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 ( / .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.

[0076] It should be understood by one of ordinary skill in the art that the therapeutic agent of the microbiome modulating composition may include a hypogravity postbiotic ferment (i.e., “hypograviotic”), a probiotic, a prebiotic, or combinations thereof. Accordingly, in other example embodiments, compositions of the present disclosure comprise less than 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 hypogravity postbiotic ferment (i.e., “hypograviotic”), 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 hypogravity postbiotic ferment (i.e., “hypograviotic”), the probiotic, and any optional prebiotics comprise about O.1 % wt / vol to about 10% wt / vol of the microbiome modulating composition.

[0077] 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, Ruminococus spp. bacteria or mixtures thereof. In certain example embodiments, the ratio of the growth of the Lactobacillus spp. bacteria to the deleterious, pathogenic bacteria, when both were applied the hypogravity postbiotic ferment (i.e., “hypograviotic”) and subjected to a growth experiment, is from about 4 to about 170, such as about 25 to about 100, such as about 35 to about 70.

[0078] 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.

[0079] In other example embodiments, the composition can be applied to treat other skin conditions, such as those associated with diaper or absorbent article wear. Thus, in some example embodiments, the species of bacteria utilized as the “mini-microbiome” used to form the hypogravity postbiotic ferments (i.e., “hypograviotics”) may be tailored to mimic a healthy diapered skin microbiome. Thus, the microbiome modulating composition can be applied to reduce diaper rash, diaper dermatitis, inflammation, or redness. In certain example embodiments, the composition can be applied to treat nasal skin irritation and other irritation of skin, epithelium, and mucous membranes.

[0080] 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.

[0081] 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, or a pad. 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.

[0082] 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.

[0083] Additional Prebiotics

[0084] In some embodiments, the microbiome modulating 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, a-cyclodextrin, [3-cyclodextrin, glucomannan, D- tagatose, 2-deoxy-D-ribose, o-methyl-D-glucoside, amylopectin, P-D-fructose, P-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, salicin, and xylitol.

[0085] Surfactant

[0086] 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.

[0087] 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.

[0088] Suitable cationic surfactants include, but are not limited to alkyl dimethylamines, alkyl amidopropylamines, alkyl imidazoline derivatives, quaternised amine ethoxylates, and quaternary ammonium compounds.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] Esters

[0093] 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.

[0094] Humectants

[0095] 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.

[0096] 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

[0097] In some embodiments, the microbiome modualting 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.

[0098] 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.

[0099] 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.

[0100] Rheology modifier

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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).

[0107] 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.

[0108] Gelling agents

[0109] In preferred example embodimets, 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) (FMC 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.

[0110] 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).

[0111] 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 urogenti al microbiome of the user.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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®.

[0116] 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 gel Ian 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 being delivered to the vaginal cavity.

[0117] 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.

[0118] 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.

[0119] Nitrogen Sources

[0120] In some embodiments, the composition may include one or more nitrogen sources to drive potential synergies. Some suitable nitrogen sources include nitrogen heterocycles, ammonia, ammonium, urea, and amino acids. 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 HCI, 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. Antimicrobial agents

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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

[0126] 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.

[0127] 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.

[0128] 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.

[0129] The present invention may be better understood by reference to the following examples.

[0130] EXAMPLES

[0131] EXAMPLE 1 : PREPARATION OF HYPOGRAVITY CONDITIONS

[0132] A hypogravity condition was formed and tested using a random positioning device. The general design of the random positioning device utilized is described in Clary, J.L., France, C.S., Lind, K.R., Shi, R., Alexander, J.S., Richards, J.T., Scott, R.S., Wang, J., Lu, X., & Harrison, L. (2022). Development of an inexpensive 3D clinostat and comparison with other microgravity simulators using Mycobacterium marinum. Frontiers in Space Technologies, doi.org / 10.3389 / frspt.2022.1032610, and Votov VV, Marovska J, Turiyski V, Ivanov SI. A new random positioning machine modification applied for microgravity simulation in laboratory experiments with rats. Inventions. 7:5 (2002) 85. doi:10.3390 / inventions7030085, which are hereby incorporated by reference. A random positioning device following a modified Clary et al. (2022) design was designed, fabricated, and assembled using 3D printing of PBS and off the shelf components.

[0133] Motion was achieved via the use of two Dynamixel MX-64 servos with overall control via a python script. Data collected for the runs is in accord with the recommendation for RPM data reporting. Axis rotation speeds for the initial trials ranged from 0.1 rpm to 3.0 rpm and were varied to ensure that the ratio of the inner and outer frame speed, y, was an irrational number. A Yost Labs 3-Space Sensor with data-loggi ng was utilized to collect x, y, z positional / orientation data and the value of the three accelerometer vectors all as a function of time. The final average value of the reduced gravity vector was calculated using the individual vectors obtained from the accelerometer according to:

[0134] Typically, tests were performed to measure < g> as a function of axis rotation speeds using the Yost Labs sensor for two hours, as at all speeds the value of the gravity vector converged to its lowest average value within that timeframe.

[0135] As demonstrated in Fig. 1 , the random positioning device could reproducibly achieve hypogravity values in the milligravity regime. For instance, setting the inner axis rotational speed to 1 .504 rpm and the outer speed to 2.4971 rpm (for a y value of 0.602298666) resulted in an average gravity value of 0.0042 x g. Rotational speeds greater than about 4 rpm began to introduce centrifugal forces which undermined the low gravity regime. As further demonstrated by Fig. 1 , hypogravity was reached within the first 5 minutes and the random positioning device was able to maintain the hypogravity condition for the entire two hours.

[0136] Further, as demonstrated in Fig. 2, the two perpendicular rotational axes (i.e., the inner and outer frames) sample all of the xyz space within the two hours. Thus, Fig. 1 and Fig. 2 demonstrate that the hypogravity conditions utilized herein are consistent during the RPM run.

[0137] EXAMPLE 2: PREPARATION OF POSTBIOTIC FERMENTS UNDER HYPOGRAVITY CONDITIONS

[0138] Freezer stocks of Lactobacillus crispatus KC18-1174-1 , Lactobacillus gasseri KC18-1132-2, Lactobacllus Iners KC17-4296-7, Enterococcus faecalls KC17-4344-5, Staphylococcus aureus KC17- 4367-2, and Candida albicans were used throughout the following examples.

[0139] Fig. 3 demonstrates the method of producing a hypogravity postbiotic ferment (i.e., "hypograviotic") as described in further detail below. Three Lactobacillus strains (L crispatus, L. gasseri, andL. iners) 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 104CFU / mL each. The chamber including MRS culture media with glucose was incubated at 37 °C anaerobically overnight until stationary. 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 including MRS media 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.

[0140] 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 MgCl2-6H20, 1mM MnCl2-4H2O, 1mM FeSO4- 7H2O, 250 piM tryptophan, 250 M cysteine, 250 M CaCI2, 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.

[0141] The bacteria subcultured on the Minimal Lactobacillus Media (MLM) media were then passaged twice while being subjected to hypogravity conditions. All passaging was done anaerobically to mimic a normal vaginal-like environment with a total culture of 200 pL and an 80 pL mineral oil recovery. During each passage, growth was performed for 48 hours in a 37 °C incubator. Between each passage, the bacteria were pelleted, washed in 2 X PBS, and resuspended.

[0142] A random positioning device, as described in Example 1 , was utilized to produce the hypogravity conditions with an average gravity value of approximately 0.004 x g .

[0143] After the two passages were performed under hypogravity conditions, the bacteria were pelleted, and the supernatant was decanted from the pellet and was recentrifuged (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 hypogravity postbiotic mixture. The gel exclusion column removed any remaining high molecular weight materials and unmetabolized glycogen.

[0144] EXAMPLE 3: EFFECTS OF POSTBIOTIC FERMENTS PRODUCED UNDER HYPOGRAVITY CONDITIONS ON BACTERIAL GROWTH

[0145] The postbiotic ferments prepared according to Example 2 were subjected to growth experiments immediately. The postbiotic ferments prepared according to Example 2 under hypogravity conditions were added to freshly cultured samples of L crispatus ("Lc”), L. gasseri (“Lg”), and L. iners (“Li”), respectively. Postbiotic ferments produced under normal gravity conditions were also prepared according to Example 2, except that the bacteria were passaged on Minimal Lactobacillus Media (MLM) culture media without being subjected to hypogravity conditions. Formation of the normal gravity postbiotic ferments are prepared according to Fig. 4. The postbiotic ferments produced under normal gravity conditions were then also added to freshly cultured samples of L crispatus (“Lc”), L. gasseri (“Lg”), and L. iners (“Li"), respectively.

[0146] The results of the L. Crispatus growth experiments are shown in Fig. 5 and are quantified below in Table 1.

[0147] Further, as a control, the growth of L. crispatus (“Lc"), L gasseri (“Lg"), and L. iners (“Li”), were also tested on growth of Minimal Lactobacillus Media (MLM) media at a pH of 5.4. The results of the control growth curve of the bacteria grown in the absence of either a hypogravity postbiotic (i.e., “hypograviotic") or a normal gravity postbiotic are demonstrated below in Table 2.

[0148] As demonstrated in Fig. 5 and Tables 1-2, the three species of Lactobacillus that were used in conjunction to produce the postbiotics all positively respond to the addition of either the normal gravity or the hypogravity postbiotic (i.e., “hypograviotic”) compared to the control sample grown in Minimal Lactobacillus Media (MLM) alone (at pH 5.4). Compared to the control sample, the addition of either postbiotic resulted in a shorter lag period, a growth acceleration during the exponential phase, and a higher final bacterial cell density. However, in each of these three areas, the hypogravity postbiotic (i.e., “hypograviotic”) was even more effective. In the case of L crispatus, the lag time went from 130 minutes in the absence of either postbiotic to 80 minutes in the presence of the normal gravity postbiotic to 40 minutes in the presence of the hypogravity postbiotic (i.e., “hypograviotic”). Final cell density (as measured by optical density at 600 nm) increased from 1 .06 in the absence of either postbiotic to 1 .25 in the presence of the normal gravity postbiotic to 1 .86 in the presence of the hypogravity postbiotic (i.e., “hypograviotic”). The exponential growth rate similarly increased from 0.006 in the absence of either postbiotic to 0.01 in the presence of the normal gravity postbiotic to 0.018 in the presence of the hypogravity postbiotic (“hypograviotic”).

[0149] To determine the effect of the hypogravity postbiotics (i.e., “hypogravitoics”) on deleterious bacteria or other organisms, samples of the hypogravity postbiotic (i.e., “hypograviotic”) and the normal gravity postbiotic were also added to freshly cultured samples of Enterococcus faecalis (“Ef), Staphylococcus aureus (“Sa”), and Candida albicans (“Ca”). Control samples of Enterococcus faecalis (“Ef’), Staphylococcus aureus (“Sa”), and Candida albicans (“Ca”) on Minimal Lactobacillus Media (MLM) media at a pH of 5.4 in the absence of a postbiotic were also tested. The results of the Enterococcus faecalis f'Ef'), Staphylococcus aureus ("Sa"), and Candida albicans (“Ca”) growth experiments are shown in Fig. 6 and Tables 1-2.

[0150] As demonstrated by Fig. 6, compared to the control sample, the addition of either postbiotic resulted in a longer lag period, a decrease in growth acceleration during the exponential phase, and a lower final bacterial cell density. However, in each of these three areas, the hypogravity postbiotic (i.e., "hypograviotic") was even more effective and best prohibited the growth of the deleterious organisms. Thus, the major effects of either postbiotic on deleterious organism growth are the opposite to those for the advantageous organisms. As demonstrated by Fig. 6, the lag phase is increased from about 1 .7 to about 3.5-fold in the presence of the hypogravity postbiotic (i.e., "hypograviotic") compared to the normal gravity postbiotic, from about 2.7 to about 5.5 fold compared to the control sample, and from about 1 .5 to about 1 .6 fold for the normal gravity postbiotic relative to the control sample. The hypogravity postbiotic (i.e., “hypograviotic”) reduced the final density of the deleterious organisms between about 3.4 to about 22-fold better compared to the normal gravity postbiotic, from about 11 .2 to about 66 fold compared to the control, and about 3.0 to about 3.3 fold for the normal gravity postbiotic relative to the control.

[0151] Table 1 : Growth variables derived from Figs. 5-6

[0152] Organism Lag Phase Exponential Final Density

[0153] (mins) Growth Rate (OD 600 nm)

[0154] Lactobacillus crispatus 40 80 0.018 0.01 1.86 1.25

[0155] Lactobacillus gaseri 42 79 0.02 0.01 1.81 1.21

[0156] Lactobacillus iners 44 81 0.02 0.01 1.70 1.22

[0157] Staphylococcus aureus 840 245 0 6e-4 0.04 0.15

[0158] Enterococcus faecalis 835 240 0 6e-4 0.05 0.17

[0159] Candida albicans 321 187 0 9e-4 0.01 0.22

[0160] ‘Hypogravity “Normal gravity

[0161] Table 2: Control growth curve quantification in the absence of a postbiotic.

[0162] Organism Lag Phase Exponential Final Density

[0163] (mins) Growth Rate (OD 600 nm)

[0164] Lactobacillus crispatus 130 6.3e-3 1.06

[0165] Lactobacillus gaseri 134 6.3e-3 1 05

[0166] Lactobacillus iners 131 6.3e-3 1.04

[0167] Staphylococcus aureus 152 1.1 e-3 0 49

[0168] Enterococcus faecalis 160 1.0e-3 0.56 Candida albicans 120 1 ,1e-3 0.66

[0169] EXAMPLE 4: EFFECT OF VARYING GRAVITY LEVELS ON POSTBIOTIC EFFICACY

[0170] Five different average values of the gravity vector < g > were then utilized to produce five different postbiotic ferments, respectively. As demonstrated by Fig. 7, the five average values of the gravity vector < g > (0 x g, 0.25 x g, 0.5 x g, 0.75 x g, and 1 .0 x g) can be maintained over the course of the growth experiment using a random positioning device as described in Example 1 . Fig. 7 demonstrates that the 1 x g curve, which indicates normal gravity, is a flat line because the RPM platform is motionless.

[0171] The postbiotic ferments were produced according to the method described in Example 2, except that the random positioning device was utilized to produce five different gravity levels: 1) 0 x g, 2) 0.25 x g, 3) 0.5 x g, 4) 0.75 x g, and 5) 1 .0 x g (i.e., "normal” gravity) which were then utilized to produce five different postbiotic ferments, respectively.

[0172] The five different postbiotic ferments were then subjected to growth experiments. The five different postbiotic ferments were respectively added to a freshly inoculated culture of / .. crispatus and growth was allowed to proceed anaerobically for 800 minutes at 37 °C. The results of the Lcrispatus growth experiments are shown in Fig. 8 and are quantified below in Table 3

[0173] Table 3: Growth variables derived from Figs. 8.

[0174] Hypogravity level Lag Phase Exponential Growth Rate Final Density

[0175] <g> (min) (AOD / min) (OD 600 nm)

[0176] As demonstrated by Table 3, the lag phase was significantly reduced, the exponential growth rate was increased, and the overall final concentration density was increased when the postbiotic ferments prepared at lower hypogravity levels were utilized.

[0177] Likewise, to determine if the effects of the postbiotic on suppressing the growth of deleterious bacteria are also dependent on the magnitude of the gravity vector used to produce the postbiotic, the five different postbiotic ferments were respectively added to freshly inoculated on S. aureus and growth was allowed to proceed anaerobically for 800 minutes at 37 °C. The results of the S. aureus growth experiments are shown in Fig. 9 and are quantified below in Table 4.

[0178] Table 4: Growth variables derived from Fig. 9

[0179] Hypogravity level Lag Phase Exponential Growth Rate Final Density

[0180] <g> (min) (AOD / min) (OD 600 nm)

[0181] 0.0 400 1.8 x 10-4 o.1

[0182] 0.25 320 8.3 x 10-4 0.2

[0183] 0.50 270 2.4 x 10-3 Q.5

[0184] 0.75 310 3.2 x 10-3 0.6

[0185] 1.0 160 4.2 x 10-3 o.7

[0186] As demonstrated by Fig. 9 and Table 4, the hypogravity-based improvement in postbiotic efficacy is not limited to a Lactobacillus-based vaginal microbiome but is a general phenomenon that can be extended to inhibiting the growth of deleterious bacteria.

[0187] EXAMPLE 5: HYPOGRAVITY POSTBIOTIC EFFECT ON NON-VAGINAL MICROBIOMES

[0188] To determine the efficacy of the hypogravity postbiotics (i.e., "hypograviotics”) on other non- vaginal microbiomes, two different postbiotic ferments were prepared as described in Example 2 under 0 x g (i.e., “hypogravity”) and 1 x g (i.e., “normal gravity”) conditions, respectively. The different postbiotic ferments were then subjected to growth experiments. The two different postbiotic ferments were respectively added to freshly inoculated cultures to a final concentration of 10% (v / v) in a culture of either Ruminococcus gnavus, a deleterious skin bacteria, for growth suppression measurements or Staphylococcus epidermidis, a beneficial skin bacteria, for growth enhancement experiments.

[0189] The results of the S. epidermidis growth experiments are shown in Fig. 10 and are quantified below in Table 5.

[0190] Table 5: Growth variables derived from Fig. 10.

[0191] Hypogravity level Lag Phase Exponential Growth Rate Final Density

[0192] <g> (min) (AOD / min) (OD 600 nm)

[0193] 0.0 85 2.8 x 10-21 5

[0194] 1.0 110 1.5 x 10-21 3

[0195] Control- no postbiotic 165 6.6 x 10'30.93

[0196] As demonstrated by Fig. 10 and Table 5, the addition of the hypogravity postbiotic (i.e., “hypograviotic”) to a culture of S. epidermidis significantly stimulates bacterial growth in all three phases of the curve. The lag phase is shortened from 165 minutes in the absence of postbiotic to 110 minutes in the presence of a normal gravity postbiotic to 85 minutes in the presence of a hypogravity postbiotic (i.e., “hypograviotic”). Final cell densities seen in stationary phase are increased from (in ODeoo units) 0.93 in the absence of postbiotic to 1 .3 in the presence of a normal gravity postbiotic to 1 .5 in the presence of a hypogravity postbiotic (i.e , "hypograviotic”). The slope of the exponential phase increases, indicating that the hypogravity postbiotic (i.e., “hypograviotic”) stimulates the growth rate of the beneficial skin bacteria.

[0197] The results of the R. gnavus growth experiments are shown in Fig. 11 and are quantified below in Table 6.

[0198] Table 6: Growth variables derived from Fig. 11.

[0199] Hypogravity level Lag Phase Exponential Growth Rate Final Density

[0200] <g> (min) (AOD / min) (OD 600 nm)

[0201] 0.0 300 5.3 x 10-4 o.21

[0202] 1.0 210 3.4 x 10-30.63

[0203] Control- no postbiotic 145 5.6 x 10’30.85

[0204] As demonstrated by Fig. 1 1 and Table 6, when a postbiotic is added to cultures of R.gnavus, the opposite effects are observed. These results are consistent with those seen in the vaginal microbiome deleterious bacterial growth experiments. The postbiotics inhibit bacterial growth with the 0 x g (hypogravity) version performing better than the 1 x g (normal gravity) formulation. Growth inhibition is observed in all three phases of growth. Lag phase increases from 145 mins in the absence of postbiotic to 210 in the presence of a normal gravity postbiotic to 300 minutes in the presence of a hypogravity postbiotic (i.e., “hypograviotic”) and the stationary phase final cell densities decrease from 0.85 in the absence of postbiotic to 0.63 in the presence of a normal gravity postbiotic to 0.21 in the presence of a hypogravity postbiotic (i.e., “hypograviotic”). The slope of the exponential phase decreases, indicating that the hypogravity postbiotic (i.e., “hypograviotic”) slows the growth rate of deleterious skin bacteria.

[0205] EXAMPLE EMBODIMENTS

[0206] Example Embodiment 1 : A method for producing a postbiotic ferment, the method comprising: preparing a culture from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof; and passaging the culture on a carbon source in a reduced gravity environment to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof; wherein the reduced gravity environment maintains a gravitational load of an average gravity value of about 0.0 x g to about 0.25 x g. Example Embodiment 2: The method of example embodiment 1 , wherein the average gravity value is from about 0.0 x g to about 0.005 x g.

[0207] Example Embodiment 3: The method as in any preceding embodiment, wherein the average gravity value is from about 0.0012 x g to about 0.0042 x g.

[0208] Example Embodiment 4: The method as in any preceding embodiment, wherein the carbon source is a glycogen carbon source.

[0209] Example Embodiment 5: The method as in any preceding embodiment, wherein the glycogen carbon source is a Minimal Lactobacillus Media (MLM) culture media without glucose.

[0210] 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.

[0211] 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.

[0212] Example Embodiment 8: 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.

[0213] Example Embodiment 9: 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 carbon source in the reduced gravity environment.

[0214] Example Embodiment 10: The method as in any preceding embodiment, further comprising pelleting the culture to form a pellet and harvesting the supernatant from the pellet to form a final postbiotic ferment.

[0215] Example Embodiment 11 : The method as in any preceding embodiment, further wherein when the postbiotic ferment is applied to a beneficial species, the beneficial species demonstrates an exponential growth lag time of about 75 minutes to about 100 minutes less than an exponential growth lag time of a beneficial species that had no postbiotic ferment applied.

[0216] Example Embodiment 12: The method as in any preceding embodiment, further wherein when the postbiotic ferment is applied to a beneficial species, the beneficial species demonstrates an exponential growth lag time of about 20 minutes to about 50 minutes less than an exponential growth lag time of a beneficial species that had a postbiotic ferment produced in a normal gravity environment applied.

[0217] Example Embodiment 13: The method as in any preceding embodiment, further wherein when the postbiotic ferment is applied to a beneficial species, a ratio of a final bacterial density (OD at 600 nm) of the beneficial species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a beneficial species without a postbiotic ferment is from about 1 .6 to about 2.0.

[0218] Example Embodiment 14: The method as in any preceding embodiment, further wherein when the postbiotic ferment is applied to a beneficial species, a ratio of a final bacterial density (OD at 600 nm) of the beneficial species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a beneficial species with a postbiotic ferment produced in a normal gravity environment is from about 1 .1 to about 1 .55.

[0219] Example Embodiment 15: The method as in any preceding embodiment, further wherein when the postbiotic ferment is applied to a deleterious species, the deleterious species demonstrates an exponential growth lag time of about 150 minutes to about 750 minutes more than an exponential growth lag time of a deleterious species that had no postbiotic ferment applied.

[0220] Example Embodiment 16: The method as in any preceding embodiment, further wherein when the postbiotic ferment is applied to a deleterious species, the deleterious species demonstrates an exponential growth lag time of about 75 minutes to about 650 minutes more than an exponential growth lag time of a deleterious species that had a postbiotic ferment produced in a normal gravity environment applied.

[0221] Example Embodiment 17: The method as in any preceding embodiment, further wherein when the postbiotic ferment is applied to a deleterious species, a ratio of a final bacterial density (OD at 600 nm) of the deleterious species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a deleterious species without a postbiotic ferment is from about is from about 0.01 to about 0.3.

[0222] Example Embodiment 18: The method as in any preceding embodiment, further wherein when the postbiotic ferment is applied to a deleterious species, a ratio of a final bacterial density (OD at 600 nm) of the deleterious species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a deleterious species with a postbiotic ferment produced in a normal gravity environment is from about 0.04 to about 0.5. Example Embodiment 19: A microbiome modulating composition comprising: a carrier; and a postbiotic ferment produced from passaging a culture of at least two of any Lactobacillus species bacteria, Limosilactobacillus species bacteria, or combinations thereof on a carbon source in a reduced gravity environment; wherein the reduced gravity environment maintains a gravitational load of an average gravity value of about 0.0 x g to about 0.25 x g.

[0223] Example Embodiment 20: The microbiome modulating composition of example embodiment 19, wherein the average gravity value is from about 0.0 x g to about 0.005 x g.

[0224] Example Embodiment 21 : The microbiome modulating composition of any preceding embodiment, wherein the average gravity value is from about 0.0012 x g to about 0.0042 x g.

[0225] Example Embodiment 22: The microbiome modulating composition of any preceding embodiment, wherein the carrier is an aqueous solution.

[0226] Example Embodiment 23: The microbiome modulating composition of any preceding embodiment, wherein the microbiome modulating composition is a gel, a liquid, a cream, a spray, or a suppository.

[0227] Example Embodiment 24: The microbiome modulating composition of any preceding embodiment, wherein the microbiome modulating composition is a vaginal insertable gel.

[0228] Example Embodiment 25: The microbiome modulating composition of any preceding embodiment, wherein the postbiotic ferment comprises about 0.1% wt / vol to about 10% wt / vol of the microbiome modulating composition.

[0229] Example Embodiment 26: The microbiome modulating composition of any preceding embodiment, wherein the carrier comprises greater than about 90% wt / vol of the microbiome modulating composition.

[0230] Example Embodiment 27: The microbiome modulating composition of 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.

[0231] Example Embodiment 28: The microbiome modulating composition of any preceding embodiment, wherein the microbiome modulating composition further comprises a prebiotic.

[0232] Example Embodiment 29: The microbiome modulating composition of any preceding embodiment, wherein the prebiotic comprises at least one Lactobacillus species bacteria-specific prebiotic, at least one Limosilactobacillus species bacteria-specific prebiotic, or combinations thereof.

[0233] Example Embodiment 30: The microbiome modulating composition of any preceding embodiment, wherein the microbiome modulating composition has a pH of from about 3.8 to about 5.0. Example Embodiment 31 : The microbiome modulating composition of 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.

[0234] Example Embodiment 32: 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 species bacteria, Limosilactobacillus species bacteria, or combinations thereof on a carbon source in a reduced gravity environment; wherein the reduced gravity environment maintains a gravitational load of an average gravity value of about 0.0 x g to about 0.25 x g.

[0235] Example Embodiment 33. The method of example embodiment 32, wherein the average gravity value is from about 0.0 x g to about 0.005 x g.

[0236] Example Embodiment 34: The method as in any preceding embodiment, wherein the average gravity value is from about 0.0012 x g to about 0.0042 x g.

[0237] Example Embodiment 35: The method as in any preceding embodiment, further comprising applying the microbiome modulating composition to a substrate, the substrate comprising a wipe or at least a portion of an absorbent article.

[0238] Example Embodiment 36: The method as in any preceding embodiment, further comprising reducing the severity of an infection or a microbial dysbiosis of the urogenital area or the skin area of the user.

[0239] Example Embodiment 37: The method as in any preceding embodiment, further comprising reducing a reoccurrence frequency of an infection or a microbial dysbiosis of the urogenital area or the skin area of the user.

[0240] Example Embodiment 38: The method as in any preceding embodiment, further comprising returning the urogenital or the skin area of the user to a microbial homeostasis.

[0241] Example Embodiment 39: The method as in any preceding embodiment, wherein the microbial dysbiosis of the urogenital or the skin area of the user includes bacterial vaginosis, candidal vaginitis ("yeast”), trichomonas vaginitis, urinary tract infections, bladder infections, or a combination thereof.

[0242] Example Embodiment 40: The method as in any preceding embodiment, wherein the microbial dysbiosis of the urogenital or the skin area of the user includes diaper rash, diaper dermatitis, inflammation, redness, or a combination thereof. Example Embodiment 41 : The method as in any preceding embodiment, wherein the microbial dysbiosis of the urogenital or the skin area of the user includes nasal skin irritation.

Claims

WHAT IS CLAIMED IS:1 . A method for producing a postbiotic ferment, the method comprising: preparing a culture from at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof; and passaging the culture on a carbon source in a reduced gravity environment to form a postbiotic ferment of the at least two of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof; wherein the reduced gravity environment maintains a gravitational load of an average gravity value of about 0.0 x g to about 0.25 x g.

2. The method of claim 1 , wherein the average gravity value is from about 0.0 x g to about 0.005 x g.

3. The method of claim 1 , wherein the average gravity value is from about 0.0012 x g to about 0.0042 x g.

4. The method of claim 1 , wherein the carbon source is a glycogen carbon source.

5. The method of claim 4, wherein the glycogen carbon source is a Minimal Lactobacillus Media (MLM) culture media without glucose.

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 of any Lactobacillus spp. bacteria, Limosilactobacillus spp. bacteria, or combinations thereof include a mixture of Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus iners.

8. 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.

9. 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 carbon source in the reduced gravity environment.

10. The method of claim 9, further comprising pelleting the culture to form a pellet and harvesting the supernatant from the pellet to form a final postbiotic ferment.

11. The method of claim 1 , further wherein when the postbiotic ferment is applied to a beneficial species, the beneficial species demonstrates an exponential growth lag time of about 75 minutes to about 100 minutes less than an exponential growth lag time of a beneficial species that had no postbiotic ferment applied.

12. The method of claim 1 , further wherein when the postbiotic ferment is applied to a beneficial species, the beneficial species demonstrates an exponential growth lag time of about 20 minutes to about 50 minutes less than an exponential growth lag time of a beneficial species that had a postbiotic ferment produced in a normal gravity environment applied.

13. The method of claim 1 , further wherein when the postbiotic ferment is applied to a beneficial species, a ratio of a final bacterial density (OD at 600 nm) of the beneficial species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a beneficial species without a postbiotic ferment is from about 1 .6 to about 2.0.

14. The method of claim 1 , further wherein when the postbiotic ferment is applied to a beneficial species, a ratio of a final bacterial density (OD at 600 nm) of the beneficial species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a beneficial species with a postbiotic ferment produced in a normal gravity environment is from about 1.1 to about 1.55.

15. The method of claim 1 , further wherein when the postbiotic ferment is applied to a deleterious species, the deleterious species demonstrates an exponential growth lag time of about 150 minutes to about 750 minutes more than an exponential growth lag time of a deleterious species that had no postbiotic ferment applied.

16. The method of claim 1 , further wherein when the postbiotic ferment is applied to a deleterious species, the deleterious species demonstrates an exponential growth lag time of about 75 minutes to about 650 minutes more than an exponential growth lag time of a deleterious species that had a postbiotic ferment produced in a normal gravity environment applied.

17. The method of claim 1 , further wherein when the postbiotic ferment is applied to a deleterious species, a ratio of a final bacterial density (OD at 600 nm) of the deleterious species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (OD at 600 nm) of a deleterious species without a postbiotic ferment is from about is from about 0.01 to about 0.3.

18. The method of claim 1 , further wherein when the postbiotic ferment is applied to a deleterious species, a ratio of a final bacterial density (OD at 600 nm) of the deleterious species with the postbiotic ferment produced in the reduced gravity environment to a final bacterial density (ODat 600 nm) of a deleterious species with a postbiotic ferment produced in a normal gravity environment is from about 0.04 to about 0.5.

19. 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 carbon source in a reduced gravity environment; wherein the reduced gravity environment maintains a gravitational load of an average gravity value of about 0.0 x g to about 0.25 x g.

20. The microbiome modulating composition of claim 19, wherein the average gravity value is from about 0.0 x g to about 0.005 x g.

21. The microbiome modulating composition of claim 19, wherein the average gravity value is from about 0.0012 x g to about 0.0042 x g.

22. The microbiome modulating composition of claim 19, wherein the carrier is an aqueous solution.

23. The microbiome modulating composition of claim 19, wherein the microbiome modulating composition is a gel, a liquid, a cream, a spray, or a suppository.

24. The microbiome modulating composition of claim 19, wherein the microbiome modulating composition is a vaginal insertable gel.

25. The microbiome modulating composition of claim 19, wherein the postbiotic ferment comprises about 0.1 % wt / vol to about 10% wt / vol of the microbiome modulating composition.

26. The microbiome modulating composition of claim 19, wherein the carrier comprises greater than about 90% wt / vol of the microbiome modulating composition.

27. The microbiome modulating composition of claim 19, 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.

28. The microbiome modulating composition of claim 19, wherein the microbiome modulating composition further comprises a prebiotic.

29. The microbiome modulating composition of claim 28, wherein the prebiotic comprises at least one Lactobacillus spp. bacteria-specific prebiotic, at least one Limosilactobacillus spp. bacteria- specific prebiotic, or combinations thereof.

30. The microbiome modulating composition of claim 19, wherein the microbiome modulating composition has a pH of from about 3.8 to about 5.0.

31. The microbiome modulating composition of claim 19, wherein the microbiome modulating composition further comprises at least one of an additional urogenital prebiotic, a surfactant, anester, a humectant, a pH adjuster, a rheology modifier, a gelling agent, a nitrogen source, and an antimicrobial agent.

32. A method for treating a 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 carbon source in a reduced gravity environment; wherein the reduced gravity environment maintains a gravitational load of an average gravity value of about 0.0 x g to about 0.25 x g.

33. The method of claim 32, wherein the average gravity value is from about 0.0 x g to about 0.005 x g.

34. The method of claim 32, wherein the average gravity value is from about 0.0012 x g to about 0.0042 x g.

35. The method of claim 32, further comprising applying the microbiome modulating composition to a substrate, the substrate comprising a wipe or at least a portion of an absorbent article.

36. The method of claim 32, further comprising reducing the severity of an infection or a microbial dysbiosis of the urogenital area or the skin area of the user.

37. The method of claim 32, further comprising reducing a reoccurrence frequency of an infection or a microbial dysbiosis of the urogenital area or the skin area of the user.

38. The method of claim 32, further comprising returning the urogenital or the skin area of the user to a microbial homeostasis.

39. The method of claim 32, wherein the microbial dysbiosis of the urogenital or the skin area of the user includes bacterial vaginosis, candidal vaginitis ("yeast"), trichomonas vaginitis, urinary tract infections, bladder infections, or a combination thereof.

40. The method of claim 32, wherein the microbial dysbiosis of the urogenital or the skin area of the user includes diaper rash, diaper dermatitis, inflammation, redness, or a combination thereof.41 . The method of claim 32, wherein the microbial dysbiosis of the urogenital or the skin area of the user includes nasal skin irritation.

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