WO2024118975A1 - Ingredient mixtures effective to modify ratios of beneficial microbial species to non-beneficial microbial species mixtures - Google Patents

Abstract

The disclosure provides methods to identify mixtures of ingredients having effectiveness in modifying a ratio of at least one beneficial microbial species to at least one non-beneficial microbial species relevant to a presence or absence of a cosmetic or cosmetic-like condition at a skin site location. The ingredient mixtures, and personal care formulations including such mixtures, are effective to impart prebiotic activity on the at least one beneficial microbial species at the skin site so as to create a shift in a human's skin site microbiome from a baseline level.

Description

INGREDIENT MIXTURES EFFECTIVE TO MODIFY RATIOS OF BENEFICIAL MICROBIAL SPECIES TO NON-BENEFICIAL MICROBIAL SPECIES TO GENERATE COSMETIC OR COSMETIC-LIKE MODIFICATIONS AT SKIN SITE LOCATIONS AND PERSONAL CARE FORMULATIONS INCLUDING SUCH MIXTURES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Patent Application No. 63/593,069, filed October 25, 2023, titled “Methods for Modifying Human Axillary Microbiota to Reduce Associated Malodorous Compounds with Prebiotic Ingredient Mixtures. ” This application also claims priority to U.S. Provisional Patent Application No. 63/454,882, filed March 27, 2023, titled “Methods for Modifying Human Axillary Microbiota to Reduce Associated Malodorous Compounds with Prebiotic Ingredient Mixtures. ” This application further claims priority to U.S. Provisional Patent Application No. 63/428,957 Filed November 30, 2022, titled “Compositions and Methods for Modifying Axillary Malodor in Humans, Including by Affecting One Or More Bacterial Species Associated Therewith with Prebiotic Materials." The disclosures of each of these provisional applications are incorporated herein in their entireties by this reference.

FIELD OF THE DISCLOUSURE

[0002] The disclosure provides methods to identify mixtures of ingredients having effectiveness in modifying a ratio of at least one beneficial microbial species to at least one non-beneficial microbial species relevant to a presence or absence of a cosmetic or cosmeticlike condition at a human skin site location. The ingredient mixtures, and personal care formulations including such mixtures, are effective to impart prebiotic activity on the at least one beneficial microbial species at the skin site so as to create a shift in a human’s skin site microbiome from a baseline level to effect a cosmetic or cosmetic-like modification thereto.

BACKGROUND OF THE DISCLOSURE

[0003] The skin is the largest human organ and the outermost interface between the human body and its environment. Many regional differences are present in a person’s skin. For instance, temperature and humidity are higher in the axillary or genital regions, approaching 37°C, the body’s core temperature, whereas skin temperatures are lower at the body’s extremities, about 30°C, and the skin tends to have less moisture in these more-exposed areas. The presence or absence of sebaceous glands and the density thereof in a skin site location influence the secretion of many lipidic compounds, including fatty acids, that contribute to the acidification of the skin pH, which varies between 4.2 and 7.9, depending on the site of measurement.

[0004] Such human biological characteristics give rise to many possibilities for creating different ecological niches housing numerous commensal bacteria, as well as fungi, viruses, archaea, and mites to create a network that varies in terms of its density and composition. Altogether, these microorganisms define the skin microbiota. A person’s skin microbiota can be associated with medical indications, such as eczema, psoriasis, etc., which typically will require treatment with therapeutics. Some skin issues, while problematic to a person who is experiencing them, may not rise to the level of needing medical interventions. Such issues are generally considered to be “cosmetic” or “cosmetic-like” in nature.

[0005] One such cosmetic or cosmetic-like issue that is a direct result of the prevalence of certain bacterial species in a human’s axillary region is an appearance of malodor. Certain bacterial species that are present in the microbiota of a human axillary region can significantly impact an intensity of a person’s underarm odor. Fresh sweat has no smell. Human underarm malodor results mainly from bacteriological activity of certain Staphylococcus sp. and Corynebacterium sp. on secretions generated in the axillary region. This means that underarm microbiota, in particular, can be considered to include a presence of certain “non-beneficial” bacterial species that function as a main influence in generation of axillary malodor generation. In this regard, apocrine secretions in the axillary region comprise long-chain fatty acids, fatty acids bound to amino acids, sulphur-containing amino acids, and hormones, which are generally too large to become volatile in their normally secreted states. However, bacterial modification of these compounds by certain bacterial species — namely, the non-beneficial bacterial species — under the typical conditions present in a human axillary region creates smaller compounds, which can become volatile and exhibit odor profiles that can be perceived as odorous.

[0006] The axillary microbiota differs from individual to individual. Depending on types and amounts of certain bacterial species present in the microbiota of a person’s axillary region, odor generated by them naturally can be highly undesirable when exhibited by an individual. Thus, even though axillary malodor is considered to be a “cosmetic” or “cosmetic-like” problem in most instances — that is, the person who exhibits even a high amount of underarm odor does not usually present with any medical issues that cause such odor — such malodor can negatively affect the mental health of a person as they may develop an insecurity due to their odor or when others may perceive their odor as offensive. As a result, a large percentage of people in industrialized countries are motivated to use personal care products having effectiveness to reduce an amount of malodor that would be present naturally in their axillary regions.

[0007] Traditional deodorant products are formulated to reduce or prevent the occurrence of axillary malodor. Deodorant products also can include fragrances to mask odor, with or without added antimicrobials. Antiperspirants, which may also include antimicrobial ingredients and/or fragrances, work to reduce or prevent sweat generated by the person which, in turn, can reduce the otherwise favorable conditions for bacterial growth in the axillary region.

[0008] Research has shown that the use of antiperspirants containing aluminum salts and, to a lesser extent, deodorant products, can affect bacterial species diversity in a person’s axillary region. It can then be inferred that these conventional products, while generally effective to prevent malodor in a relevant user population in context, might also result in problematic changes to a user’s axillary microbiota. For example, if one is left with higher bacterial diversity resulting from product use, the bacterial species that return can create a more unpleasant smell if those increased species are those associated with the formation of malodorous compounds. That is, if the bacterial species that return with antiperspirant and/or deodorant use include a higher ratio of non-beneficial bacterial species to beneficial bacterial species, it can be the case that conventional deodorant and antiperspirant products may make a person’s axillary malodor worse than it would be natively.

[0009] Another “cosmetic” or “cosmetic-like” problem that can be associated with an excess of certain microbiota in the scalp region is redness, irritation, dryness, and/or flaking. The fungal genus Malassezia is highly abundant on the scalp, with M. restricta being implicated as a primary cause of dandruff and other conditions often associated with an “unhealthy” scalp region in a person. The scalp microbiota is also composed of many of the same bacterial species found in the axillary and other regions of the skin, where some of these are “beneficial” and “non-beneficial” in the context of a specific human body site location. For example, the amounts of the “beneficial” scalp bacterial species Staphylococcus sp. can differ both between “healthy” and “non-healthy” scalps, and even between different areas on the same scalp. While the scalp microbiota is a complex and sometimes delicate community of microorganisms that can vary from person to person, current knowledge supports a finding that an appearance of dandruff or dandruff-like conditions in a person may result from a degree of dysbiosis — or misbalance of naturally occurring microbiota — in a person’s scalp.

[0010] Notably, personal care products formulated for scalp treatment for conditions associated with scalp redness, irritation, dryness, and/or flaking have primarily been formulated to address the presence of fungal genus Malassezia, without regard to the other naturally occurring microbiota that may have a beneficial effect when present in a person’s scalp. Current treatments directed toward improvement in dandruff or dandruff-like conditions are not designed to address a need to maintain a balance of the various microbiota that can affect the overall health of a person’s scalp. In other words, as currently formulated, personal care products formulated for the scalp region can result in a dysbiosis of the scalp region. In some people, dandruff treatments can cause skin irritation, redness, and/or irritation as a side effect, even while the fungal origins of dandruff-like conditions may otherwise be fully addressed.

[001 1] As illustrated above, existing methods to address the conditions of axillary malodor and dandruff-like conditions, while effective to address the most prevalent aspects as appearing cosmetically in a person — namely, malodor and fungus-caused redness, irritation, dryness, and/or flaking — none of these existing treatments appear to directly address what may be the root causes of these common skin disorders.

[0012] A promising approach to imparting cosmetic modifications associated with the microbiota present in or associated with various skin site locations is to evaluate the effects of certain beneficial and non-beneficial microbiota on a person’ s cosmetic condition at that skin site. While research on the skin microbiome lags years behind studies of the gut microbiome, there is now emerging evidence that microbial imbalances, or dysbiosis, on the skin could play a role not just in body odor but in other cosmetic or cosmetic-like conditions, such as dandruff, dry skin, etc. Moreover, although research is only just developing as to what effects the various microbiota may impart to a person’s skin site location, it can be inferred that a “healthy” or “intact” skin microbiome could be relevant to a person’s skin health, or even more broadly, their overall health. At a minimum, it could be expected that indiscriminate reduction or elimination of the amounts and types of microbial species present in a person’s skin location when treating a cosmetic or cosmetic-like condition may not be the optimum approach for a product type that is used by most people on a daily basis. [0013] Like with the gut microbiome, various approaches have been proposed to modify baseline ratios of microbiota that may be present in a person’s skin site location, whether as natively occurring or after use of products that might have affected the natural skin microbiota. One approach used for modification of the gut microbiome is the use of nutrients that can preferentially feed the “good” — or “beneficial” — microorganisms that are natively present in a person’s skin site location to increase an amount of these microorganisms, with a goal of providing a cosmetic or cosmetic-like modification to a skin site location to a person in need thereof. In this process, prebiotics have been proposed to stimulate the growth of specific benefit-promoting microbes in a skin location. A prebiotic is an ingredient that imparts bioselective activity to a microbiota element with a goal of generating a beneficial effect of interest. In contrast to other microbiome modifications, there can be several advantages to this method. First, unlike with probiotic treatments, there is no need to apply living bacteria; thus, there is a reduced chance of a skin immune reaction. Moreover, prebiotics currently in use today are typically well-defined compounds for which side effects are well studied. The INCI names and safety sheets are normally available. There are also disadvantages, however. As indirect methods, the results can be hard to track using existing techniques to impart prebiotic activity to skin site locations from personal care formulations. Imprecise use of prebiotics on skin could also stimulate non-targeted bacterial species that natively occur in lesser abundance in a skin site location. In short, the effect of prebiotics can be unpredictable given the variability in the skin microbiome, physiology, and immune response in different individuals. Moreover, a prebiotic ingredient that demonstrates relevant activity in vitro, may otherwise not be effective to modify relevant skin microbiota when delivered from a personal care formulation suitable for use at that skin location.

[0014] In view of the above, there is a need for improvements in methods to provide cosmetic or cosmetic-like modification to a human’ s skin site location without also causing undesirable side effects resulting from an unintended reduction in an amount of beneficial microbial species. There is a need to identify and apply methods to identify and use cosmetically appropriate ingredients selected to reduce an amount of non-beneficial microbial species that can be implicated in causing undesirable cosmetic or cosmetic-like conditions in a human skin site location. There is also a need to identify and apply methods to identify cosmetically appropriate ingredients having utility in promoting the growth of beneficial microbial species in a skin site location, while not at the same time also promoting the growth of non-beneficial microbial species. For example, there is a need for prebiotic materials suitable for use in a personal care formulation that can function to shift a ratio of beneficial microorganisms to non-beneficial microorganisms in a human axillary region, a scalp region, or other skin site locations in need of improvement in a cosmetic or cosmetic-like condition when a person uses the formulation at a relevant location over a period of time. The present disclosure provides this and other benefits.

SUMMARY OF THE DISCLOSURE

[0015] Disclosed are methods of identifying an ingredient mixture effective to modify a cosmetic or cosmetic-like condition in a skin site location, and personal care formulations containing the ingredient mixture. The methods involve selecting a skin site location of interest for imparting a cosmetic or cosmetic-like modification thereto, identifying at least one beneficial microbial species and at least one non-beneficial microbial species as relevant to providing the modification thereto by effecting a change in the ratio of beneficial microbial species to non-beneficial microbial species as compared to a baseline level after treatment with the ingredient mixture comprising prebiotic activity on the at least one beneficial species.

[0016] Significantly, it has been determined that single ingredients do not generate suitable effectiveness in generating the desired shifting of a ratio of beneficial microbial species to non-beneficial microbial species, where the ratio is determined to be relevant to a presence or absence of a cosmetic or cosmetic-like condition at a skin site location. In this regard, it has been determined that mixtures comprising at least two ingredients identified from a two-step high throughput screening methodology wherein human review and selection/deselection of ingredients are important to confer the desired cosmetic or cosmeticlike modification to a skin site location. In the two-step high throughput screening methodology, individual microbial species having relevance to a cosmetic or cosmetic-like condition associated with a skin site location can be identified for screening against a large number of chemical compounds, nutrients, and/or growth conditions. Because of the large amount of data generated from this initial screen, and to improve predictability of the performance of such ingredients, supervised learning methods have been initially identified as relevant to the analysis of data derived from the high throughput screening herein. For instance, a machine learning model trained to differentiate ingredient effectiveness on individual bacterial species and to predict specificity to each bacterial species can be used to analyze the data and make predictions about which mixtures are suitable. Such predictions are reviewed by a human expert for appropriateness for use in a personal care formulation. [0017] After generation and analysis of the results of the first high throughput screening procedure, a second high throughput screening is conducted to identify chemical compounds, nutrients, and/or growth conditions that not only are likely to affect the growth of one or more microbial species present in a skin site location that are relevant to a cosmetic or cosmetic-like condition of interest thereon, but also to generate information that can be used to provide an associated cosmetic or cosmetic-like modification to a skin site location in a person in need thereo.

[0018] In this second high throughput screening step, chemical compounds and growth conditions can be selected for further testing. Such selected compounds and conditions can be identified as actually or potentially affecting the growth of microbial species present in or associated with a human skin location where a presence or absence of cosmetic or cosmeticlike condition may also be directly or indirectly influenced by an amount of such microbial species at that location. In this regard, ingredients found to affect the growth of beneficial and non-beneficial microbial species, are analyzed for appropriateness for use in personal care formulations suitable for use in a skin site location. That is, while a chemical compound, nutrient, and/or growth condition may be shown in the first high throughput screening step to be effective to affect the growth of one or more relevant microbial species, such test parameters will still need to be validated to ensure that they are, in fact, appropriate for use in the context of a product intended for use on human skin or scalp areas.

[0019] Also disclosed are compositions containing mixtures of ingredients effective for modifying a cosmetic or cosmetic-like condition in a skin site location. The Examples demonstrate the efficacy of these compositions.

[0020] It is to be understood that the summary above is provided to introduce a simplified selection of concepts that are described further in the detailed description. It is not meant to identify key or essential features of the claimed subject matter, the scope of which is defined uniquely by the claims that follow the detailed description. Furthermore, the claimed subject matter is not limited to implementations that solve any disadvantages noted above or in any part of this disclosure. BRIEF DESCRIPTION OF THE FIGURES

[0021] FIGS. 1A-1D are bar graphs showing the effectiveness of 4% (w/w) Blend 83 (FIGS. 1A and IB) and 4% (w/w) Blend 50 (FIGS. 1C and ID) on Mock Axillary Bacterial Species Community applied from Hybrid Cosmetic Stick.

[0022] FIG. 2 is a bar graph showing the effects of Blend 83 added at 0.4% to Sweat-Like Media (SLM) on the amount of thiol produced versus the controls-SLM only and 1% NaHCO₃.

[0023] FIG. 3 is a bar graph showing differences between various levels of 4% (w/w) Blend 83 between and among the baseline amounts of S. epidermidis and Corynebacterium sp. in non-incubated SLM (“Start”) incorporating MOP and HOP mock microbial communities and incubated SLM (“Finish”) having MOP and HOP mock microbial communities. The results are compared against NaHCO-,, as a control odor reducing composition.

[0024] FIGS. 4A and 4B are line graphs showing the effect of 4% (w/w) Blend 83 in Hybrid Cosmetic Stick on “non-Beneficial” Axillary Bacterial Species (FIG. 4A) and in anhydrous Stick Formulation on “Beneficial” Axillary Bacterial Species (FIG. 4B).

[0025] FIGS. A and 5B are bar graphs showing axillary bacterial species differential abundance (FIG. 5A) and Corynebacteria sp. differential abundance (FIG. 5B) with and without 4% (w/w) Blend 83 from Hybrid Cosmetic Stick in five human volunteers and Day 0 to Day 5. FIG. 5C is a line graph with two panels showing analysis of Corynebacteria sp. differential abundance with and without 4% (w/w) Blend 83 in Hybrid Stick Formulation Day 0 to Day 5.

[0026] FIG. 6A is a line graph with two panels showing single volunteer volatile organic compound GC/MS analysis with and without 4% (w/w) Blend 83 from Hybrid Cosmetic Stick. FIG. 6B is a bar graph showing volatile organic compound amount differences in human volunteer between left and right axillary regions.

[0027] FIG. 7 is a bar graph showing volatile organic compound amount differences in human volunteers (n=8) between left and right axillary regions. Only compounds with >25% reduction shown.

[0028] FIG. 8 is a line graph showing trans-epidermal water loss (“TEWL”) analysis on human volunteers (n = 33) with 4% Blend 83 in aqueous solution.

[0029] FIG. 9 is a bar graph showing a GC/MS analysis of 3M2H-related compound reduction with Blend 2 compared with Blend 83 and control. [0030] FIGS. 10A-10D are bar graphs showing GC/MS analyses of relative thiol production with Blend 2 compared with Blend 83 and control.

[0031] FIG. 11 is a bar graph showing the specificity of Blend 83 and Blend 2 on Mock Community comprising non-beneficial and beneficial axillary bacterial species.

[0032] FIG. 12 is a schematic showing metabolic dye screening procedure for effect of test and control ingredients on Mock “Dandruff’ Bacterial Species Community.

[0033] FIG. 13 is a bar graph showing the effect of ingredient mixtures on in vitro Mock “Dandruff’ Microbial Species Community.

[0034] FIG. 14 shows pie charts of consumer perception study for Blends 15, 16, and 162 in shampoo base vs. shampoo base only.

[0035] FIG. 15 is a bar graph showing metabolic activity study of 1% blend 162 with and without salicylic acid (SA) compared to controls of over-the-counter dandruff actives.

[0036] FIGS. 16A and 16B are column graphs showing volunteer perception study for 1% blend 162 delivered from shampoo base A: bacterial sequencing data (FIG. 16A) and Malassezia sequencing data (FIG. 16B).

[0037] FIG. 17 is a column graph showing volunteer perception study for 1% blend 162 delivered from shampoo base A: squalene and squalene monohydroperoxide ratio.

[0038] FIG. 18 shows panels with images from volunteer perception study for 1 % blend 162 delivered from shampoo base A: dermascope images.

DETAILED DESCRIPTION OF THE DISCLOUSURE

[0039] While the specification concludes with claims that particularly point out and distinctly claim the invention, it is believed the present invention will be better understood from the following description.

[0040] The present disclosure can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well any of the additional or optional ingredients, components, or limitations described herein.

[0041] All percentages, parts and ratios are based upon the total weight of the compositions of the present invention, unless otherwise specified. All such weights as they pertain to listed ingredients are based on the active level and, therefore do not include carriers or by-products that may be included in commercially available materials.

[0042] The components and/or steps, including those which may optionally be added, of the various implementations of the present invention, are described in detail below. [0043] All ratios are weight ratios unless specifically stated otherwise.

[0044] All temperatures are in degrees Celsius, unless specifically stated otherwise.

[0045] Except as otherwise noted, all amounts including quantities, percentages, portions, and proportions, are understood to be modified by the word “about”, and amounts are not intended to indicate significant digits.

[0046] As used herein, an element or step recited in the singular and proceeded with the word “a” or “an” should be understood as not excluding plural of said elements or steps, unless such exclusion is explicitly stated. Furthermore, references to “one implementation,” “one aspect,” or “one embodiment” of the present disclosure are not intended to be interpreted as excluding the existence of additional implementations, aspects, or embodiments that also incorporate the recited features.

[0047] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.”

[0048] As used herein, the terms “about,” “approximate,” and “at or about” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, or ±5% of the specified value, e.g., about 1” refers to the range of 0.8” to 1.2”, 0.8” to 1.15”, 0.9” to 1.1”, 0.91” to 1.09”, 0.92” to 1.08”, 0.93” to 1.07”, 0.94” to 1.06”, or 0.95” to 1.05”, unless otherwise indicated or inferred. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0049] Any ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. Such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0. 1 % to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the subranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and Tess than z’. Likewise, the phrase ‘x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In some aspects, the term “about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase “about x’ to ‘y’” includes “about ‘x’ to about ‘y’”.

[0050] The term “substantially” is meant to permit deviations from the descriptive term that do not negatively impact the intended purpose. All descriptive terms used herein are implicitly understood to be modified by the word “substantially,” even if the descriptive term is not explicitly modified by the word “substantially.”

[0051 ] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0052] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the various methods and materials suitable for use with the various inventions disclosed herein are now described. Functions or constructions well-known in the art may not be described in detail for brevity and/or clarity.

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0054] All publications and patents cited in this specification are herein incorporated by reference as if each individual publications or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publications by virtue of prior disclosure. Further, the publication dates provided could be different from the actual publication dates that may need to be independently confirmed. [0055] This written description uses Examples to disclose various aspects one or more inventions, including the best mode, and also to enable a person of ordinary skill in the relevant art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those of ordinary skill in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

[0056] As used herein, “skin site location” means any surface of a human’s body that is populated by a collection of microbial species that are “beneficial” and “non-beneficial” in relation to a cosmetic or cosmetic-like condition at that body site location, where that location would be of interest for imparting of an improvement thereto by imparting a shift in the abundance of the microbial species from a baseline level, as discussed in detail herein. As would be appreciated, in the U.S., a “cosmetic” is legally defined as a product that is used for cleansing, beautifying, promoting attractiveness, or altering the appearance of a person. As used herein, a “cosmetic” or “cosmetic-like” modification would be associated with a change in a feature of a person’ s skin site condition that is consonant with this definition of “cosmetic.”

[0057] A “beneficial microbial species” is a biological element in a collection of microbial species — that is, the “microbiota” — that are present in or associated with a human’s skin site location. Such beneficial skin microbial species are present in a first amount natively in a person asa baseline amount. In accordance with the disclosure herein, the beneficial skin microbial species can experience an increase from the baseline amount with an ingredient mixture that exhibits prebiotic activity on the beneficial skin microbial species. The change in the amount of beneficial skin microbial species from a baseline amount to a different amount is associated with a modification in a cosmetic or cosmetic-like condition at the human skin site location. In some aspects, a human skin site location of interest for imparting a cosmetic or cosmetic-like modification thereto can comprise at least one beneficial skin microbial species present in or associated with the location. In further aspects, a human skin site of interest for imparting a cosmetic or cosmetic-like modification thereto can comprise two or more beneficial skin microbial species present in or associated with the location.

[0058] A “non-beneficial microbial species” is a biological element in a collection of microorganisms that are present in or associated with a human’s skin site location that can be associated with an undesirable or unwanted cosmetic or cosmetic-like condition. In some aspects, a skin site location comprises at least one non-beneficial microbial species natively present in or associated with the location. In further aspects, a skin site location comprises two or more non-beneficial microbial species natively present in or associated with a skin site location.

[0059] In the present disclosure, the term “baseline” can be used to describe both of a baseline or native level of microbial species present in a person’s skin site location at a point after the person has refrained from using a personal care formulation at that location for a period of time associated with a “washout” of the effects that were intended to be imparted by such personal care formulations. An appropriate time to assess a baseline level of beneficial and non-beneficial microbial species in a specific skin site location after a washout period can vary. The washout period needed for a person to see a desired cosmetic or cosmetic-like modification can vary according to the person’s individual microbial species make up at their skin site location, as well as in relation to the type of personal care formulations that they may have used prior to use of the personal care formulations having effective amounts of the ingredient mixtures herein. For example, a baseline level of beneficial to non-beneficial microbial species in a person’ s axillary region can be determined after that person has not used antiperspirant or deodorant products for a washout period of about 3, 7, 10, 14, or more days.

[0060] It should be noted that a microbial species that is “non-beneficial” when present in greater amounts in a skin site location may not necessarily be “non-beneficial” when present at the same location at lower amounts. For example, lower amounts of C. jeikeium and S. hominis in a person’ s axillary region may not result in noticeable axillary odor, and the person may not then be associated with an odor that is typically characterized with “axillary malodor.” But when these microbial species are present in the person’s axillary region at higher levels, an amount and type of odor that is typically associated with “axillary malodor” may be noticeable. It should therefore be understood that identification of a microbial species as “beneficial” or “non-beneficial” at a specific skin site location is in relation to a presence of amount thereof that can be directly or indirectly associated with the presence or absence of a cosmetic or cosmetic-like condition that is noticeable by a person and, therefore, can be of interest in imparting a cosmetic or cosmetic-like modification thereto.

[0061] Moreover, a microbial species can be “non-beneficial” in one skin site location, but “beneficial” in other location. For example, C. acnes is typically considered to be “non- beneficial” when present on a person’s facial areas, but “beneficial” when present in a scalp location. It follows that in addressing modifications to any amounts and ratios of beneficial to non-beneficial microbial species from a baseline level, a specific skin site location is relevant. [0062] When relevant beneficial and non-beneficial skin microbial species at a skin site location comprise different bacterial species, the bacterial species can be “commensal skin bacteria” in that they reside natively on the skin of most humans without causing negative health effects in the human host. However, as discussed herein, certain commensals can cause undesired cosmetic conditions, such as malodor, when the skin site location is an axillary region. In other body site locations, the non-beneficial skin microbial species can comprise non-bacterial species, for example fungal species that can be associated with an undesirable cosmetic or cosmetic-like condition in a skin site location that is the scalp area. [0063] “Microbial abundance” means what amount of a skin site location microbiome is made up of a specific microbial species. A change in microbial abundance resulting from the ingredient mixtures herein is measured in relation to a baseline determination, as discussed further herein.

[0064] “Prebiotic” means any substance or combination of substances that may be utilized as a nutrient by a microorganism, may induce the growth and/or activity of a microorganism, may induce the replication of a microorganism, may be utilized as an energy source by the microorganism, and/or may be utilized by the microorganism for the production of biomolecules (i.e., RNA, DNA, proteins, primary and secondary metabolites). In accordance with the disclosure herein, a prebiotic is effective in promoting the growth of beneficial microbial species that is relevant with a skin site location of interest as compared to a baseline level. A prebiotic ingredient mixture having utility in the methodology herein can be determined by the results of individual ingredient screening and associated analysis thereof as set out hereinafter. While ingredients that are determined to increase an amount of growth of one or more beneficial microbial species relevant to or associated with a skin site location may already be identified in the prior art as comprising some type of prebiotic activity, the methodology of the present disclosure can provide validation of that prebiotic activity and/or information associated with an amount of prebiotic ingredient or conditions that are more or less beneficial for a specific microbial species. The methodology herein can also identify ingredients, and associated amounts and conditions related thereto, that were previously unknown to possess any prebiotic activity for the beneficial microbial species associated with or relevant to a human skin site location.

[0065] “Non-prebiotic” means that an ingredient exhibits an effect of not promoting growth, slowing and/or reducing the growth of a microbial species associated with or relevant to a skin site location of interest as compared to a baseline level. In accordance with one or more objectives of the present disclosure, an ingredient that exhibits “non-prebiotic” activity is not always the same category of materials that would be characterized as “anti-microbial,” in that the latter material is intended to kill or otherwise inactivate microbes of interest, of one or more microbial species. An non-prebiotic ingredient having utility in the methodology herein can be determined by the results of ingredient screening as set out hereinafter. While ingredients that are determined to decrease an amount of growth of one or more microbial species relevant to or associated with a skin site location may already be identified in the prior art as comprising some type of non-prebiotic activity, the methodology of the present disclosure can provide validation of that non-prebiotic activity and/or information associated with an amount of non-prebiotic ingredients or suitable conditions that are more effective to reduce the growth of a specific non-beneficial microbial species. The methodology herein can also identify ingredients, and associated amounts and conditions related thereto, that were previously unknown to possess any non-prebiotic activity for the non-beneficial microbial species associated with or relevant to a human skin site location.

[0066] “Metabolism” means any chemical reaction occurring inside a microorganism. Metabolism includes anabolism, the synthesis of the biological molecules (e.g., protein synthesis and DNA replication) and catabolism, the breakdown of biological molecules. [0067] “Increase” means increases above baseline levels, or as compared to a control. [0068] “PCR” means polymerase chain reaction and includes real-time PCR, quantitative PCR (“QPCR”), semi-quantitative PCR, and combinations thereof.

[0069] “Personal care formulation” means a composition suitable for topical application on mammalian skin and/or other keratinous tissue such as hair, scalp, and nails. “Topical” means the surface of the skin or other keratinous tissue. “Personal care formulation” as used herein can include sticks, roll-ons, sprays, pump-sprays, aerosols, soap bars, powders, solutions, gels, creams, balms, and lotions. As set out further herein, a suitable personal care formulation from which a selected ingredient mixture can be delivered to provide a desired cosmetic or cosmetic-like modification to a skin site location resulting from a change in a baseline ratio of beneficial to non-beneficial microbial species thereon is configured with ingredients that substantially do not interfere with an amount of prebiotic activity provided by the selected ingredient mixture. Moreover, suitable personal care formulations according to the disclosure herein also substantially do not include ingredients other than the selected prebiotic ingredient mixture that increase or decrease a ratio in the beneficial microbial species to non-beneficial microbial species in the skin site location. In this regard, the personal care formulations having utility herein are configured to impart a change in a ratio of the beneficial microbial species to non-beneficial microbial species present in a skin site location from a baseline level substantially only from an effectiveness of a selected ingredient mixture to impart prebiotic activity on at least one beneficial microbial species and, optionally, non-prebiotic activity on at least one non-beneficial microbial species present in the skin site location. To this end, a change in a ratio of beneficial microbial species to non- beneficial microbial species at the skin site location from a baseline level is substantially not provided by any prebiotic and/or non-prebiotic activity imparted from other ingredients present in the personal care formulations from which the prebiotic and/or non-prebiotic activity is delivered from incorporation of a selected ingredient mixture therein. In other implementations, the personal care formulations comprise ingredients (other than the selected ingredient mixture) that are identified as providing substantially no prebiotic activity on the beneficial microbial species at that skin site location. Yet further, the ingredients in the personal care formulation (other than the selected ingredient mixture) are selected to substantially not impart any non-prebiotic activity on the non-beneficial microbial species at the skin site location.

[0070] “Increase in relative amount of beneficial microbial species to non-beneficial microbial species” means that a ratio of beneficial microbial species to non-beneficial microbial species at the skin site location at a first time prior to application of a personal care formulation comprising an effective amount of an ingredient mixture selected as having effectiveness to increase a growth of the at least one beneficial microbial species in the skin site location is less than a corresponding ratio of beneficial microbial species to non- beneficial microbial species after the formulation has been applied to the skin location for a period of time at an indicated frequency, for example at least once daily. An increase in the ratio of beneficial microbial species to non-beneficial microbial species as generated from the methodology herein is sufficient to provide a desired cosmetic or cosmetic-like modification to generate an improvement in a cosmetic or cosmetic-like condition at the skin site location. For example, use of a personal care formulation comprising an effective amount of an ingredient mixture selected as having a prebiotic effect on beneficial axillary region activity can reduce an amount of axillary malodor in a person who uses the formulation daily for a period of time. Moreover, that lesser amount of axillary malodor can be maintained as long as the person continues to use the formulation regularly.

[0071 ] “Cosmetic or cosmetic-like modification” in the context of a skin site location means regulating and/or improving a person’ s skin condition as compared to a baseline condition via application thereto of a personal care formulation comprising an effective amount of a selected ingredient mixture as set out in more detail herein. A non-limiting example of a relevant cosmetic or cosmetic-like modification provided by the methodology herein comprises a reduction in an amount of malodor at a body site location, such as in an axillary region or foot area. Further cosmetic or cosmetic-like modifications as used herein includes improving skin appearance by reducing redness and/or improving consistency of coloration along length or in an area of a person’s skin surface, and/or by providing a smoother, more even surface at a skin site location, such as by reducing an amount of surface roughness. A reduction in trans epidermal water loss (“TEWL”) as determined by one or more methods can also comprise a desired cosmetic or cosmetic-like modification herein. Another cosmetic or cosmetic-like modification to a person’s scalp area, where the scalp is a “skin site location” of interest for imparting a modification thereto. In this regard, a cosmetic or cosmetic-like modification to a skin site location that is a scalp can be associated with regulating and/or improving a person’s scalp condition. Nonlimiting examples of modifying a scalp condition can include improving scalp appearance by reducing an amount of redness, a reduction in a feeling of dryness or tightness, and/or reducing an appearance of flakiness or skin sloughing that can cause itching or other undesirable cosmetic conditions in a person’s scalp area. Skin can also be a cosmetic or cosmetic-like condition of interest for modification. Similarly, redness on an exposed skin area, such as the forearm or legs, can be undesired to the person who exhibits this condition. Slight physical discomfort at a body site location, such as irritation, itchiness or tightness, can also be a cosmetic or cosmetic-like condition of interest for imparting a cosmetic or cosmetic-like modification thereto at a skin site location.

[0072] “Substantially free of” refers to about 2% or less, about 1% or less, or about 0.1% or less of a stated ingredient. “Free of” refers to no detectable amount of the stated ingredient or thing.

[0073] “Effective amount” means an amount of a subject ingredient mixture that is high enough to provide a significant positive modification of the cosmetic or cosmetic-like condition to be treated in a person’s hair or skin location, namely, a desired effect of modifying a ratio of beneficial microbial species to non-beneficial microbial species in a skin site location from a baseline amount in a person in need of a cosmetic or cosmetic-like modification, where that modification is directly or indirectly related to the relative amounts of beneficial microbial species to non-beneficial microbial species present in or associated with the location. An effective amount of an ingredient mixture having prebiotic activity in relation to at least one beneficial microbial species present in a person’ s skin site location can vary with the particular cosmetic or cosmetic-like condition being treated, the severity of the condition, the duration of the treatment, the nature of concurrent treatment, and like factors. As discussed hereinafter, a candidate ingredient mixture generated from the screening of different chemical compounds, nutrients, and growth conditions as affecting the growth of one or more beneficial or non-beneficial microbial species can be “effective” when used in neat form but may not be as effective when incorporated in a personal care formulation. [0074] In accordance with the disclosure herein, a personal care formulation that is effective to generate a cosmetic or cosmetic- like modification in a human skin site location where such modification is associated with a change in a baseline ratio of beneficial and non-beneficial microbial species will include an effective amount of an ingredient mixture where a majority of the prebiotic activity imparted by the mixture can be delivered from the personal care formulation to a person’s skin site location in an actual use scenario. “Majority” in relation to an amount, such as in relation to prebiotic activity on at least one beneficial microbial species present in or associated with a skin site location, means more than 50%.

[0075] “Dermatologically acceptable” means that the compositions or components thereof so described are suitable for use in contact with human keratinous tissue without undue toxicity, incompatibility, instability, allergic response, and the like.

[0076] “Cosmetically acceptable,” as used herein, means that the compositions, formulations or components described are suitable for use in contact with human keratinous tissue without undue toxicity, incompatibility, instability, allergic response, and the like. All compositions described herein which have the purpose of being directly applied to keratinous tissue are limited to those being cosmetically acceptable.

[0077] “Apply” or “application” as used in reference to a personal care formulation, means to apply or spread the compositions of the present invention onto a skin site location such as the person’s skin or scalp regions.

[0078] “Rinse-off’ means the intended product usage includes application to skin and/or hair followed by rinsing and/or wiping the product from the skin and/or hair within a few seconds to minutes of the application step. The product is generally applied and rinsed in the same usage event, for example, a shower.

[0079] "Leave-on,” in reference to personal care formulations, means formulations intended to be applied to and allowed to remain on skin and scalp in actual use case scenarios. These leave-on formulations are to be distinguished from formulations, which are applied to the skin or scalp and subsequently (in a few minutes or less) removed either by washing, rinsing, wiping, or the like. Leave-on formulations thus exclude rinse-off applications such as shampoos, rinse-off conditioners, facial cleansers, hand cleansers, body wash, or body cleansers. The leave-on formulations may be substantially free of cleansing ingredients. For example, "leave-on formulations” may be left on a skin site location for at least 15 minutes or even longer depending on the use and formulation type. [0080] In the context of the present disclosure, a human skin site location of interest for imparting a cosmetic or cosmetic-like modification thereto can comprise at least one beneficial microbial species and at least one non-beneficial body site microbial species, where the meaning of “beneficial” and “non-beneficial” are in accordance with that skin site location. A ratio of beneficial skin microbial species to non-beneficial skin microbial species at a skin site location at a first time in a baseline amount can be increased by contacting the location with a personal care formulation comprising an amount of prebiotic activity effective to increase or enhance the growth of a least one beneficial skin microbial species present in or associated with the skin site location. In significant aspects, the personal care formulation comprises an ingredient mixture selected from a plurality of ingredient mixtures, where the selected ingredient mixture exhibits prebiotic activity on at least one beneficial skin microbial species present in or associated with that skin site location. The ingredient mixture can also impart prebiotic activity to more than one beneficial microbial species present in that skin site location when delivered in an effective amount from suitable personal care formulations. The ingredient mixture can also impart a non-prebiotic effect to at least one non-beneficial microbial species present at that body site location.

[0081] A goal or objective resulting from use of a selected ingredient mixture in a personal care formulation configured for use at the body site location is a generation of a change in the ratio of beneficial microbial species to non-beneficial body site microbial species in a person’s skin site location as compared to a baseline ratio thereof, where the change in ratio generates a desired cosmetic or cosmetic-like modification at or associated with that skin site location.

[0082] A further goal or objective resulting from the use of a selected ingredient mixture is a maintenance of an improvement to cosmetic or cosmetic-like condition in the person’ s skin site location, where such maintenance can be directly or indirectly associated with a continued elevated ratio of beneficial to non-beneficial microbial species at the skin site location. In this regard, the present disclosure can allow a cosmetic or cosmetic-like improvement to be imparted to the skin site location as long as the person uses a personal care formulation incorporating an effective amount of an ingredient mixture selected to impart prebiotic activity to at least one beneficial microbial species present at that skin site location. In other words, if the person uses the personal care formulation comprising an effective amount of prebiotic activity for one or more beneficial microbial species for a period of time, they will continue to experience an improved cosmetic or cosmetic-like condition associated with use of that formulation, as compared to a baseline condition. [0083] In broad constructs, the disclosure provides methods enhancing the abundance of at least one beneficial bacterial species at or associated with a human skin site location of interest such that a ratio of an amount of the at least one beneficial microbial species and at least one non-beneficial body site microbial species where the ratio is modified from a baseline ratio thereof to a ratio that is associated with a desired cosmetic or cosmetic-like modification at the skin site. In some aspects, the ratio change can be provided by the increase in abundance of the at least one beneficial microbial species along with an attendant reduction of abundance of at the least one non-beneficial microbial species present therein. In other aspects, an increase in the growth of at least one beneficial microbial species can result in an increase in the ratio of that microbial species even when the growth of the at least one non-beneficial microbial species at the skin site location substantially does not change.

[0084] In significant implementations, the methods of modifying a ratio of the at least one beneficial microbial species to the at least one non-beneficial body site microbial species present in or associated with a selected skin site location can be provided by a mixture of two or more ingredients, in which at least one of the ingredients in the mixture imparts prebio tic activity on the at least one beneficial microbial species. Still further, at least one ingredient in the mixture exhibits prebiotic activity on the at least one beneficial microbial species present in or associated with a selected skin site location and at least one ingredient in the mixture exhibits non-prebiotic activity on the at least one non-beneficial body site microbial species at the location. In further implementations, the ingredient mixture comprises two or more ingredients, where at least one of the ingredients exhibits prebiotic activity on at least one beneficial microbial species present in or associated with the selected skin site location and at least one of the ingredients exhibits non-prebiotic activity on the at least one non-beneficial body site microbial species at the location.

[0085] It was hypothesized that the enhancement of the abundance of at least one beneficial microbial species present in or associated with a selected skin site location as a result of the prebiotic activity imparted by an ingredient mixture could result in a decrease in an amount of at least one non-beneficial microbial species. Such non-beneficial microbial species reduction could result from an activity imparted by the beneficial microbial species thereon. In other words, an increase in abundance of at least one beneficial microbial species at the skin site location could indirectly provide a cosmetic or cosmetic-like modification to the skin site location that results from a non-prebiotic activity imparted on the non-beneficial microbial species by the beneficial microbial species. Using an example of S. epidermidis as the at least one beneficial microbial species in a skin site location, at least one ingredient in an ingredient mixture can exhibit prebiotic activity on this beneficial bacterial species, and the resulting growth can generate some non-prebiotic — or more specifically, “lantibiotic” — effect on an associated non-beneficial body site microbial species at the axillary region. It would then follow that an incorporation of an ingredient mixture imparting such lantibiotic activity from S. epidermidis on a non-beneficial microbial species, a ratio of the at least one beneficial microbial species and the at least one non-beneficial body site microbial species can be modified from a baseline level by application of the formulation to the skin site location.

[0086] For clarity, in regard to an effectiveness of the ingredient blends herein, the present disclosure addresses a change in a ratio of at least one beneficial microbial species to at least one non-beneficial microbial species present in or associated with a selected human skin site location, where the total amounts of each of the at least one beneficial microbial species and at least one non-beneficial microbial species, as measured by a ratio, are different after application of a personal care formulation comprising an effective amount of ingredient mixtures to that body site location from a first time to a second time. To this end, the ingredient mixtures having effectiveness herein are selected for incorporation in a personal care formulation not only for their effectiveness in changing a ratio of an amount at least one beneficial microbial species to at least one non-beneficial microbial species present in or associated with a selected skin site location, but also any activity to be conferred on the respective species by the ingredient mixtures are deliverable from a personal care formulation configured for application to the skin site location. Put another way, if an ingredient mixture is effective to modify a ratio of the at least one beneficial microbial species to at least one non-beneficial microbial species as present in or associated with a selected skin site location in testing, but that activity is not deliverable from a suitable personal care formulation, the mixture will have little, if any, utility in generating a desired cosmetic or cosmetic-like modification at the skin site location. Accordingly, a relevant aspect of the present disclosure is confirmation of an effectiveness of an ingredient mixture on changing a ratio of the at least one beneficial microbial species to the at least one non-beneficial microbial species present in or associated with the skin site location as delivered from a personal care formulation suitable for cosmetic use at the location.

1 [0087] Initial information about an effect of individual ingredients on microbial species abundance that are present in human skin surfaces can be derived from information generated in a series of high throughput screening tests, as further discussed hereinafter. In this regard, individual ingredients that may or may not have utility in generating ingredient mixtures — also called “blends” herein — can initially undergo testing to assess an efficacy thereof in affecting the growth of specific beneficial microbial species that can be natively present in one or more skin site locations. From testing of the individual ingredients, a plurality of ingredient mixtures can be generated for in vitro and in vivo testing on collections of beneficial and non-beneficial microbial species present in or associated with a skin site location of interest for imparting of a cosmetic or cosmetic-like modification thereto. Such in vitro and in vivo testing can be configured to generate information from which a presence or absence of a desired cosmetic modification can be generated in a real-life human when an ingredient mixture incorporating an effective amount thereof is used at the skin site location from a first time to a second time.

[0088] Ingredient mixtures that are shown to have efficacy in enhancing the abundance of the at least one beneficial microbial species and in reducing or moderating the abundance of the at least one non-beneficial microbial species as relevant to a human skin site location can be incorporated in a suitable personal care formulation, where that personal care formulation can be tested for a suitability as a delivery vehicle for the ingredient mixture to the skin site location in a real-life use case. Once the viability of a personal care formulation configuration to suitably impart prebiotic activity to the at least one beneficial microbial species present in or associated with the skin site location, with or without non-prebiotic activity on at least one non-beneficial microbial species therein, the personal care formulations can be configured with an effective amount of a selected ingredient mixture to deliver a desired cosmetic or cosmetic-like modification to the selected body site in a real-life human who uses the personal care formulations from a first time to a second time.

[0089] A notable aspect of the present disclosure is a differentiation from prior approaches to imparting prebiotic activity to skin microbial species. In this regard, some prior methodologies are disclosed in U.S. Patent No. 9,271,924 (“the ‘924 patent”) and U.S. Patent Publication No. 20220192949, the disclosures of which are incorporated herein in their entireties by this reference. In each of these disclosures, the approach is to identify individual ingredients that are effective in imparting prebiotic activity to certain bacterial species, where such ingredients can be incorporated in personal care formulations. The underlying functional premise of these approaches is that identification of prebiotic activity on a microbial species can be translated into effectiveness in a personal care product when an ingredient is included therein. In contrast, and as described in detail herein, the inventors have determined that improvements in imparting a cosmetic or cosmetic-like modification can be achieved by use of ingredient mixtures comprised of more than one ingredient, where such mixtures collectively provide an effect to modify a ratio of beneficial to non-beneficial microbial species present at the skin site location.

[0090] By way of explanation, it was hypothesized that a combination of ingredients may be more effective to impart a cosmetic or cosmetic-like modification to a skin site location when such modification was associated with microbial species present at that location. In this regard, it was thought that there would likely to be different nutritional needs for various microbial species present at each location at least because different beneficial and non- beneficial microbial species are present in various skin sites in a human. Moreover, even if the same microbial species may be located at different locations, those species are likely to be present in different amounts at various locations. It was thus hypothesized that imparting of a cosmetic or cosmetic-like modification to a specific skin site location could be facilitated by a combination of ingredients, some of which may serve as a nutrient source for beneficial microbial species, whereas other ingredients may operate to decrease or moderate the growth of non-beneficial microbial species present in or associated with a skin site location. By “tuning” an ingredient mixture that could promote the abundance of beneficial microbial species while at the same time reducing the abundance of non-beneficial microbial species at that same location, it was hypothesized that improved results in conveying cosmetic or cosmetic-like benefits could be obtained.

[0091] Rather than approaching a cosmetic or cosmetic-like condition that might be associated with amounts and types of microbial species at a skin site location as a single ingredient/microbial species problem, the inventors endeavored to impart prebiotic activity on beneficial microbial species while still recognizing that the presence of non-beneficial microbial species could also be beneficial to the skin site location. Put another way, the inventors determined that it was not only better to increase an amount of beneficial microbial species in a skin site location, but that the non-beneficial microbial species should also be considered when selecting ingredient mixtures having relevant prebiotic activity in the context of a specific skin site location. The approach to modification of a cosmetic or cosmetic-like condition at a skin site location is thus considered to be a multi-factorial problem, as opposed to selecting for use a single ingredient that has effectiveness to promote the abundance of a single beneficial microbial species or to reduce or stop the growth of a non-beneficial microbial species.

[0092] In this regard, different areas of a human skin surface may typically be populated by the same microbial species, but with different skin locations including different groupings and ratios of microbial species, some of which may be beneficial microbial species in one location, but non-beneficial microbial species at another. Even when a skin site location may be populated by the same microbial species, the amounts thereof may or may not be associated with a presence or absence of a cosmetic or cosmetic-like condition. It follows that an ingredient mixture that works on a collection of microbial species that may be present in amounts that are “beneficial” and “non-beneficial” at one skin site location, may nonetheless be present in amounts that are not “beneficial” and “non-beneficial” at other skin site locations. The inventors thus have approached the problem of imparting a cosmetic or cosmetic-like modification for a condition that is directly or indirectly associated with a presence of a type and amount of certain microbial species as needed in the context of a specific skin site location and the condition of interest. In this regard, the processes comprise identification of the types of microbial species affecting the presence or absence of a cosmetic or cosmetic-like condition, assessing the amounts of the individual microbial species as might affect the condition, and generating a collection of ingredients that together are configured to impart a relevant change in the amounts of microbial species when the mixture is included in an effective amount in a personal care formulation configured for the skin site location. A result of the change in a ratio of the amounts of the microbial species is to impart the desired cosmetic or cosmetic- like condition to the skin site location.

[0093] Moreover, and as set forth in the Examples hereinafter, generation of a cosmetic or cosmetic-like modification is not necessarily achieved when materials that have known prebiotic activity are included in an ingredient mixture having a collection of ingredients. For example, addition of yeast extract — as a known skin prebiotic material — does not impart a prebiotic effect to an ingredient mixture that otherwise imparts non-prebiotic activity to beneficial microbial species in a human axillary region. The desired modification to a cosmetic or cosmetic-like condition to be imparted by an ingredient mixture when the mixture is incorporated in an effective amount in a personal care formulation configured for a skin site location is imparted by the collection of ingredients, as opposed to an individual effectiveness of each ingredient independently. It further follows that the ingredient mixture should substantially not contain any ingredients that impart non-prebiotic activity on the at least one beneficial microbial species at the skin site location at least because addition of a prebiotic ingredient cannot be expected to increase an amount of the beneficial microbial species if that species has been reduced or eliminated by one or more ingredients in an ingredient mixture in the first order. Or, put another way, addition of a prebiotic material, such as yeast extract, may not promote abundance of a microbial species that has otherwise substantially been reduced or removed from skin site locations during treatment.

[0094] An aspect of the present disclosure comprises identifying at least two microbial species having relevance to a human skin site of interest for generation of a desired cosmetic or cosmetic-like modification thereto. In this regard, ingredient mixtures effective to provide a desired cosmetic or cosmetic- like modification can be selected from a plurality of generated ingredient mixtures when use thereof generates a change in a ratio defined by an amount of the at least one microbial species identified as “beneficial” to an amount of the at least one microbial species identified as “non-beneficial,” where the ratio change is measured from a baseline level determined at a first time. The cosmetic or cosmetic-like modification to a skin site location can be effective to generate a desired improvement to cosmetic or cosmetic-like condition to a human in need thereof. In this regard, human skin microbial species (or subspecies) that are typically present at a skin site location can be identified for modification of starting amounts thereof at a baseline, where the modification is facilitated by activity of the ingredient mixtures on the growth of the beneficial microbial species by an imparting of prebiotic activity on such beneficial microbial species.

[0095] As would be understood, human skin includes a large variety of microbial species that are typically present in one or more skin site locations. While many microbial species can be present between and among various skin site locations on a human, typically only certain microbial species can be implicated in a presence of an undesirable cosmetic or cosmetic-like condition, such as axillary malodor or dandruff. As a first step to identifying and selecting ingredient mixtures having effectiveness in modifying a ratio of at least one beneficial microbial species to at least one non-beneficial microbial species in a specific skin site location, the disclosure provides identification and selection of a plurality of microbial species that are present in a skin site location of interest for modification thereof from a baseline ratio so that a desired cosmetic or cosmetic-like modification can be imparted thereto when a suitable personal care formulations incorporating an effective amount of a selected ingredient mixture is used for a period of time. [0096] In accordance various implementations of the present location, each of a plurality of microbial species typically present in or associated with a specific human skin site location that can be relevant to or associated with a desired cosmetic or cosmetic-like modification can be identified as a “beneficial microbial species” or a “non-beneficial microbial species” in the context of the skin site location of interest. A collection of microbial species (and subspecies) can be selected for modification of a baseline amount thereof based on their prevalence at one or more skin site locations and in relation to whether a priori knowledge exists indicating that they are implicated in one or more cosmetic conditions of interest for modification in a human in need thereof.

[0097] Table 1 presents an example collection of bacterial species and subspecies typically present on human skin that can be selected as microbial species having relevance to a specific skin site location. Such bacterial species can be “beneficial” or “non-beneficial” in different skin site locations as related to whether they can be associated with an undesirable cosmetic or cosmetic-like condition at a specific location.

[0098] TABLE I: Bacterial Species Typically Present on Human Skin

Bacterial Species

Cutibacterium granulosum

Micrococcus luteus

Staphylococcus epidermidis

Roseomonas mucosa

Corynebacterium jeikeium

Cutibacterium acnes

Staphylococcus capitis Corynebacterium tuberculostearicum

Staphylococcus aureus Staphylococcus hominis

Malassezia restricta

Malassezia globosa

[0099] In various aspects, a skin site location of interest for imparting a cosmetic or cosmetic-like modification thereto can be selected and at least one beneficial microbial species and at least one non-beneficial microbial species can be identified as relevant to providing the modification thereto by effecting a change in the ratio of beneficial microbial species to non-beneficial microbial species as compared to a baseline level after treatment with an ingredient mixture comprising prebiotic activity on the at least one beneficial species. The selected skin site location can comprise one of a human axillary region, a human scalp area, a face region, an arm region, a trunk region, a leg region, a foot region, a hand region, a genital region, or any other skin site location that may be of interest for effecting a cosmetic or cosmetic-like modification thereto. A selected skin site location can be characterized according to a type of beneficial and non-beneficial microbial species present in or associated with that skin site.

[0100] As an example, a human axillary region can be selected as the skin site location of interest and a collection of one or more beneficial microbial species and non-beneficial microbial species associated with that location can be identified as being of interest for imparting a cosmetic or cosmetic-like modification thereto. The cosmetic or cosmetic-like modification made thereto can be provided by an ingredient mixture that comprises prebiotic activity effective to enhance the growth of at least one of the beneficial microbial species at the axillary skin site. A sample collection of beneficial and non-beneficial bacterial species relevant to the imparting of a cosmetic or cosmetic-like modification to a human axillary region are shown in Table 2 below.

[0101 ]TabIe 2: Sample Collection of Human Axillary Beneficial and Non-Beneficial Bacterial Species [ [

[ Beneficial ]

Micrococcus luteus ■ Beneficial [ [ Corynebacterium tuberculostearicum i Non-Beneficial i

[0102] At least one of the above microbial species denoted as “beneficial” and at least one denoted as “non-beneficial” in the context of a presence in an amount directly or indirectly associated with a cosmetic or cosmetic-like condition of interest for modification at the skin site location can be selected for assessment of an effectiveness of an ingredient mixture in affecting the growth of the at least one beneficial microbial species. An effectiveness of an ingredient mixture in imparting a cosmetic or cosmetic-like modification to an axillary skin site location by modifying a ratio of beneficial microbial species to non-beneficial microbial species is set out in the Examples hereinafter. In an illustrative example, the at least one axillary beneficial microbial species can comprise S. epidermidis and the at least one axillary non-beneficial microbial species can comprise C. jeikeium, when the latter is present at a level at which axillary malodor can be noticeable to the person or to another person. An ability of an ingredient mixture to increase a ratio of the S. epidermidis to the C. jeikeium at the axillary skin site location from a baseline level can be generated by providing prebiotic activity of the mixture on the S. epidermidis and, optionally, providing non-prebiotic activity on the C. jeikeium. The change in the ratio of S. epidermidis to the C. jeikeium at the skin site location can be associated with an ability of an effective amount of the mixture to impart a desired cosmetic or cosmetic-like modification when incorporated into a personal care product configured for use in the human axillary region when the composition is suitable to confer at least the prebiotic activity to the S. epidermidis present in a real-life human’ s axillary area. In other implementations, the beneficial axillary microbial species can comprise S. epidermidis and Cutibacterium acnes and the non-beneficial axillary microbial species can comprise C. jeikeium, C. tuberculostearicum, and S. hominis, when the latter species are present in an amount in the skin site location at which a characteristic axillary malodor can be noticed by the person or someone else.

[0103] As would be appreciated, in the context of a human axillary region, a desired cosmetic or cosmetic-like modification to be conferred by a personal care formulation including an ingredient mixture having effectiveness to modify a ratio of at least one beneficial microbial species to non-beneficial microbial species from a starting or baseline ratio is most likely to be a reduction in an amount of axillary malodor in the human from use of a personal care formulation comprising an effective amount of the ingredient mixture. As shown in the Examples hereinafter, a baseline amount of axillary malodor can be shown by a starting amount of body site microbial species identified as “non-beneficial” in context, for example, C. jeikeium, as compared to a starting amount of S. epidermidis as the beneficial microbial species. A baseline amount of malodor can be determined after the human stops using deodorant and/or antiperspirant products for a period of time, that is, after a “washout period,” as defined hereinabove. As would be appreciated, the human can achieve a reduction in an amount of axillary malodor resulting from use of a personal care formulation including an effective amount of a selected ingredient mixture without first stopping using an antiperspirant and/or deodorant products for a period of time, however, a baseline level of malodor for that person can more effectively be determined when the person’s axillary region is substantially free of active ingredients present in products configured to reduce an amount of perspiration and or axillary malodor.

[0104] In a further non-limiting example, a scalp region can comprise the skin site location of interest and a collection beneficial and non-beneficial bacterial species can be identified as relevant to a desired cosmetic or cosmetic-like condition associated with the scalp region when at least such “non-beneficial” microbial species are present in an amount at which the person’s scalp exhibits redness, irritation, dryness, and/or flaking. An example of such categorization is illustrated below in Table 3.

[0105] Table 3: Scalp Skin Site Beneficial and Non-Beneficial Microbial Species

] Microbe [ Group [

\Staphy lococcus epidermidis Beneficial [

[ Cutibacterium acnes [ Beneficial ]

[0106] A plurality of beneficial microbial species and/or non-beneficial microbial species can be selected for modification of a ratio of a total amount of beneficial microbial species and non-beneficial microbial species in a human scalp region from a baseline amount, where such baseline amount can be directly or indirectly associated with a presence of a cosmetic or cosmetic-like condition of interest for modification so as to impart a cosmetic or cosmeticlike benefit to the person. The modification of the ratio of beneficial microbial species to non- beneficial microbial species by an ingredient mixture having prebiotic activity to increase the abundance of the beneficial microbial species and, optionally, to reduce the abundance of the non-beneficial microbial species can be inferred to be associated with a cosmetic or cosmetic - like modification that can be provided to a person’s scalp region when they use a personal care formulation comprising an effective amount of the ingredient mixture for a period of time sufficient to impart the desired modification. As noted, a cosmetic or cosmetic-like modification can comprise or be associated with a reduction an amount of scalp redness, itchiness and/or flakiness from a baseline amount. In an example, the plurality of scalp beneficial microbial species can comprise one or more of .S', epidermidis, C. acnes, M. luteus, and C. granulosum and the non-beneficial microbial species can comprise one or more of M. restricta and S. capitis. More broadly, the non-beneficial microbial species can comprise the fungal genus Malassezia with or without S. capitis. Again, the baseline amount of each of these microbial species can be determined after a washout period, as discussed previously.

[0107] It should be noted that these beneficial microbial species and non-beneficial microbial species as potentially relevant to a human scalp region can be defined as a heretofore unrecognized collection of bacterial species having utility in assessing the relative growth of beneficial microbial species in relation to non-beneficial microbial species that may be present in a human’s scalp area natively. In this regard, this grouping of beneficial microbial species and non-beneficial microbial species can have utility in determining an effectiveness of a prebiotic material in increasing an amount of growth of at least one beneficial microbial species and, in some aspects, the assessment of an attendant a decrease in growth of at least one non-beneficial microbial species that is natively present in a human’ s scalp area. Accordingly, the collection of microbial species in the listing above can also have utility in determining the effectiveness of active ingredients that have or may have utility in claim validation and/or substantiation of scalp-care products.

[0108] A ratio modification in an amount of beneficial microbial species to non-beneficial microbial species resulting from prebiotic activity of an ingredient mixture can be associated with the imparting of a desired improvement in a cosmetic or cosmetic-like condition to a person’s scalp area when an effective amount of a selected ingredient mixture having activity to enhance the growth of the beneficial microbial species relative to an attendant amount of the non-beneficial microbial species is incorporated into a personal care product configured for application to a scalp area. In this regard, a generated change in the ratio can be in relation to a difference from a baseline level after the personal care formulation has been used by the person for a period of time sufficient to impart a desired cosmetic or cosmetic-like modification relevant to a person’s scalp area. To this end, a desired cosmetic or cosmeticlike modification to a person’s scalp area can comprise one or more of a reduction in redness, itchiness, dryness, and/or flakiness in their scalp area. More generally, a modification of a ratio of at least one beneficial microbial species to at least one non-beneficial microbial species in a human scalp area after use of a scalp-effective ingredient mixture as delivered from a suitable personal care product can be perception by a user that their scalp is “healthier.” As would be appreciated, daily or almost-daily use of the personal care formulation can enable the beneficial cosmetic or cosmetic-like benefit to be imparted to the person’s scalp area to be continued for as long as the person uses the formulation.

[0109] As with the axillary skin site location example, in a scalp use case, the baseline ratio of the at least one beneficial microbial species and the at least one non-beneficial microbial species can be determined after a suitable washout period of at least about 3 days, or 5 days, 7 days or longer in which the person does not use any personal care products. However, a desired cosmetic or cosmetic-like modification can be obtained even when the person does not undergo a washout period prior to use of a scalp formulation comprising an effective amount of an ingredient mixture having appropriate prebiotic activity on the at least one beneficial microbial species and at least one non-beneficial microbial species thereon.

[01 10] Yet further, a cosmetic or cosmetic-like condition associated with a skin site location can be selected as being of interest for modification and a ratio of at least one beneficial microbial species and to at least one non-beneficial microbial species can be associated with a presence or absence of that condition, where the designation of a species as “beneficial” or “non-beneficial” are in relation to a presence of a cosmetic or cosmetic-like condition of interest for modification, as discussed previously. As would be appreciated, different skin site locations can be associated with various desirable and undesirable cosmetic or cosmetic-like conditions, such as an appearance of odor, irritation, redness, dryness, flakiness etc.

[01 1 1] In other implementations, a skin site location can be identified using one or more characteristics, where such characteristics can be directly or indirectly influenced by a ratio of at least one beneficial and at least one non-beneficial microbial species present in or associated with that location. In relation to a skin site location having a cosmetic or cosmeticlike condition present in or associated therewith, one or more microbial species can be identified as “beneficial” and one or more microbial species can be identified as “non- beneficial” in relation to a skin condition type or category, when such microbial species are present at the location in an amount that directly or indirectly affects the presence of the condition. For example, a skin site location can be identified as “dry,” “moist,” “sebaceous,” “non-sebaceous” or any other categorization where a collection of beneficial and non- beneficial microbial species can be identified as typically being associated with the type of category. In accordance with the disclosure therein, a desired cosmetic or cosmetic-like modification can be to change the type or category of the skin location from a first category to a second category. For example, modification of a ratio of beneficial microbial species to non-beneficial microbial species in a skin type or category of “dry” to a different skin type or category of “non-dry” or “normal” can be imparted by the methodology herein.

[01 12] As reported by Byrd, et al. (Byrd, Allyson L., Yasmine Belkaid, and Julia A. Segre, "The human skin microbiome." Nature Reviews Microbiology, 16.3 (2018): 143-155.), the following list of bacterial species are recognized as associated with various skin location types or categories:

[01 13] Table 4: Skin site characteristics and bacterial species associated therewith (from Byrd, et. al)

§ Alar crease, cheek, glabella,

Nare, antecubital external auditory fossa, inguinal canal, manubrium, crease, interdigital retroauricular

* Hypothenar palm, web, popliteal crease, occiput, volar forearm fossa back II Toe web space, toenail, plantar heel [01 14] In some implementations, one or more of the ingredients in a selected ingredient mixture can exhibit prebiotic activity on the at least one beneficial microbial species present in or associated with a skin site type or category, where the prebiotic activity is effective to increase a ratio of the at least one beneficial microbial species to at least one non-beneficial microbial species as present in the location. The identification of a microbial species as “beneficial” or “non-beneficial” can be as associated with a cosmetic or cosmetic-like condition where such designated bacterial species directly or indirectly affect the presence or absence of the condition. As set out herein, the increase is measured from a baseline level generated at a first time to a second time after the skin site location is treated with an effective amount of the ingredient mixture. Yet further, an increase in growth of a microbial species designated as “beneficial” in context can be directly or indirectly related to a generation of a desired cosmetic or cosmetic-like modification at the skin site location when a suitably configured personal care formulation incorporating an effective amount of an ingredient mixture having prebiotic activity on at least one beneficial microbial species is used by the person in need of the cosmetic or cosmetic-like modification.

[01 15] In further implementations, one or more ingredients in an ingredient mixture can exhibit non-prebiotic activity on the at least one microbial species designated as “non- beneficial” at the skin site location in the context of a presence or absence of a cosmetic or cosmetic-like condition of interest, where the non-prebiotic activity is effective to not promote, to reduce or to otherwise moderate the growth of the non-beneficial microbial species at the location when the ingredient mixture is delivered from a suitably configured personal care product. Such decrease in abundance of the at least one non-beneficial microbial species can be directly or indirectly related to the generation of a desired cosmetic or cosmetic-like modification at the selected skin site location when a personal care formulation incorporating an effective amount of the prebiotic component is used by a person at the skin site location.

[01 16] As noted previously, while prebiotic activity has been understood to have beneficial effects when incorporated in nutritional supplements and other digestible products, to date, there has been little progress in the development of prebiotic materials having proven effectiveness in providing desired cosmetic or cosmetic-like modifications delivered from topical application of personal care formulations configured for use in skin and/or scalp locations. In order to address this lack of progress, the inventors herein approached the problem in a heretofore unrecognized systematic approach in which high throughput screening method was used to evaluate the effect of a large number of ingredients and conditions on a collection of microbial species that were determined to be associated with various locations on the human skin surface, where such microbial species were identified as known to have or potentially having a direct or indirect effect on the presence or absence of one or more cosmetic or cosmetic-like conditions relevant to human skin surfaces.

[01 17] To this end, it was hypothesized by the inventors herein that ratios of beneficial microbial species and non-beneficial microbial species could have relevance to a presence or absence of a cosmetic or cosmetic-like condition associated with a skin site location. Thus, modification of such a ratio in context could have relevance to imparting a cosmetic or cosmetic-like modification at a specific skin site location. To be effective in generating a large enough dataset to elucidate what might be relatively small changes in vivo as needed to impart a desired change in a cosmetic or cosmetic-like condition at a skin site location, high throughput screening of microbial activity was selected as an approach to identify potentially relevant chemical compounds, nutrient sources, and bacterial growth conditions on a plurality of microbial species as might be relevant to a variety of skin site locations. In the context of the present disclosure, results obtainable from high throughput screening methods can provide an initial understanding of a biochemical interaction or possible role of a chemical compound, nutrient, and/or growth condition in generating a change in an amount of growth of a bacterial species associated with a cosmetic or cosmetic-like condition that is of interest for modification thereof at one or more skin site locations.

[01 18] As would be understood, high throughput screening assays comprise automated methods that allow for a large number of chemical compounds, nutrients, and growth conditions to be rapidly evaluated for a specific type of bioactivity at the molecular or cellular level. This approach can have utility in identifying compounds, nutrients, and growth conditions that might modulate specific biological pathways — here the growth rates and relative abundances of microbial species having a direct or indirect relevance to a presence or absence of a cosmetic or cosmetic-like condition present in or associated with a skin site location — were initially developed by pharmaceutical companies for drug discovery. High throughput screening has also been used in Synthetic Biology applications. As would be understood, Synthetic Biology is an emerging field of science that involves redesigning organisms for useful purposes by engineering them to have new abilities. High throughput processes have also been developed to efficiently screen for bacterial and anti-fungal activity to address an increasing need for new actives to address new and/or resistant bacterial and fungal diseases.

[01 19] Given these recent improvements in high throughput screening methods to measure bacterial activity, it was hypothesized that a variation of existing high throughput methodologies used for antibiotic and antifungal drug discovery processes might have utility in identifying chemical compounds and cosmetically relevant conditions may exhibit not only prebiotic activity on beneficial microbial species, but that would also not promote, reduce — or at least moderate — the growth of non-beneficial microbial species in a skin site location of interest.

[0120] A previous high throughput process to identify prebiotic activity on skin commensal bacterial species was disclosed in US Patent No. 9,271,924, previously incorporated by reference. However, that method addressed the identification of single ingredients that exhibited a prebiotic effect on individual bacterial species that are typically present on the skin or forearm. The ‘924 Patent did not recognize that in a skin surface environment where both beneficial microbial species and non-beneficial microbial species are present in a ratio that can be directly or indirectly associated with a presence or absence of a cosmetic or cosmetic-like condition of interest for modification in a skin site location, more than one ingredient, for example two or more, may be more useful to generate the desired modification thereto.

[0121] In the context of the high throughput screening method used herein, the inventors hypothesized that prebiotic activity as needed to improve an amount of beneficial microbial species in the context of a skin site location for use in personal care formulations configured for delivering a cosmetic or cosmetic-like modification directly or indirectly derived from a change in an amount of microbial species relevant to a cosmetic or cosmetic-like condition could be preliminarily identified from data derived from a suitably configured high throughput microbial activity screening method. By taking a whole skin surface approach to a selection of bacterial species that are known to be associated with a presence or absence a cosmetic or cosmetic-like condition in a human, it was thought that enriched information about what chemical compounds, nutrients, and growth conditions could affect a ratio of beneficial microbial species to non-beneficial microbial species in a specific skin site location could better be determined. This enriched information could then be used to identify ingredient mixtures that might deliver enhanced cosmetic or cosmetic-like conditions to a human when delivered from a suitably configured personal care formulation. That is, from the results of high throughput screening, individual chemical compounds, nutrients, and/or growth conditions could be identified from which to generate mixtures of ingredients for additional testing for prebiotic activity on at least one beneficial microbial species and at least one non-beneficial microbial species present in a skin site location as relevant in the context of a presence or absence of cosmetic or cosmetic-like condition.

[0122] Also, given the unpredictability associated with both the prediction of and validation of prebiotic activity as relevant to the variety of microbial species present on human skin, the inventors understood that it would likely be necessary to confirm the effectiveness of an ingredient mixture predicted to have prebiotic activity by human expert analysis of the high throughput results in order to develop personal care formulations relevant to cosmetic product applications. In this regard, the inventors recognized that it would not be enough to take the “best performing” chemical compounds, nutrients, and growth conditions combine these to generate a suitable personal care formulations for use in a human. Instead, it would be necessary to evaluate the data generated from the high throughput screening process to identify those chemical compounds, nutrients, and growth conditions that would be appropriate in the context of cosmetic product that could be used on a human skin surface.

[0123] Further, it was of interest to confirm that a system-generated ingredient mixture would also demonstrate effectiveness when delivered from suitable personal care formulation. Unlike in the ‘924 Patent and U.S. Patent Publication No. 20220192949 — also previously incorporated by reference herein — it was recognized by the inventors herein that an ingredient mixture that was effective to modify a range of beneficial microbial species to non-beneficial microbial species in a skin site location would nonetheless have little value if it could not be delivered from a personal care formulation in an amount effective to impart a desired cosmetic or cosmetic-like modification to a skin site location in need thereof.

Moreover, any personal care formulations in which a selected ingredient mixture having prebiotic activity would also need to be in an acceptable format so that consumers would be motivated to use the products.

[0124] In regards to the high throughput screening process used herein, a large number of chemical compounds, nutrients, and growth conditions can be assessed in the high throughput screening method, where such compounds, nutrients, and growth conditions can be derived from literature sources, such as a list of materials that have previously been determined to impart prebiotic activity on microbial species that have been identified as beneficial in one or more skin site locations. For example, yeast extract and inulin are currently understood to exhibit prebiotic effect on several bacterial species, and such ingredients can be incorporated for testing in the high throughput screening.

[0125] Moreover, and as would be appreciated, given the cosmetic and cosmetic-like applications for which the present disclosure is directed, it can be beneficial to focus on chemical compounds, nutrients, and/or growth conditions that are more likely to have relevance to skin care applications. In this regard, chemical compounds that are generally recognized as safe (“GRAS”) materials can also be included for an initial high throughput screening. Similarly, chemical compounds that have assigned TNCI names (“International Nomenclature Cosmetic Ingredient”) can be incorporated in the screening method for a presence or absence of prebiotic activity on the plurality of bacterial species tested in the high throughput screening. As would be understood INCI names are systematic names internationally recognized to identify cosmetic ingredients, such as plant extracts, oils, and chemicals. They are developed by the International Nomenclature Committee and published in the International Cosmetic Ingredient Dictionary and Handbook. Ingredient lists derived from the expert knowledge of the inventor team can also be generated.

[0126] As an initial aspect of designing the high throughput screening methodology, individual microbial species having relevance to a cosmetic or cosmetic-like condition associated with a skin site location can be identified for screening. Given a goal of generating as many data points as possible in an initial high throughput screening process, there can be a preference of first being over inclusive of chemical compounds, nutrients, and/or growth conditions, even when some of these test parameters may not be natively associated with utility in skin care formulations. For example, some chemical compounds potentially having activity vis a vis the growth of microbial species being tested in the high throughput screening may be hard to procure in commercially relevant amounts or are not suited for use in cosmetics; nonetheless, information derivable from their use in a screening process may provide useful information that can allow more appropriate ingredients to be identified for further testing.

[0127] Both microbial species that could be identified as “beneficial” and “non-beneficial” were included in the screening. As discussed previously, “beneficial” and “non-beneficial” designations are made in the context of a cosmetic or cosmetic-like condition of a specific skin site location, as not all microbial species impart the same influences in the same amounts at different skin site locations. Also, because there can be substantial overlap between different microbial species at different skin site locations, a comprehensive approach was taken to evaluate as many potentially relevant microbial species as practicable in the high throughput screening protocols so as to generate a large number of data points that could have utility in generating effective ingredient mixtures for modification of various cosmetic or cosmetic-like conditions.

[0128] Information of whether a particular chemical compound, nutrient, and/or growth condition was relevant to increase or decrease the growth of a microbial species known to affect— or that was hypothesized to likely affect — a cosmetic or cosmetic-like condition associated with one or more skin site locations was generated from the high throughput screening. In this regard, information generated about a prebiotic or non-prebiotic effect of an ingredient on a microbial species was developed in the high throughput screening for further evaluation for chemical compounds, nutrients, and/or growth conditions that could be useful to enhance the growth of microbial species identified as “beneficial” in the context of one or more skin site locations, nutrients, and growth conditions. Such microbial growth information could also have utility when that same microbial species is identified as a “non-beneficial” when present at or associated with a different skin site location at various amounts.

[0129] As a non-limiting example, the collection of microbial species used in the high throughput screening method can comprise the list of microbial species in Table 1 hereinabove or the more specific list of bacterial species as set out in Table 2. Other microbial species, such as one or more fungal species identified as directly or indirectly causing or influencing a presence or absence of a cosmetic or cosmetic-like skin condition, such as those set out in Table 3 hereinabove, can be selected for screening.

[0130] As would be appreciated, a high throughput screening method having utility to identify chemical compounds, nutrients’ and/or growth conditions can be configured to assess the effect thereof on the metabolic activity of each of the individual microbial species under the test conditions. High throughput screening assays can be run for a range of chemical compounds, nutrients, and/or growth conditions on microbial species metabolic activity to produce information representing the relationship between test parameters and prebiotic or non-prebiotic activity for each microbial species included in the test sample.

[0131] In an exemplary high throughput screening methodology, a metabolic assay can be used to assess the effect of a plurality of chemical compounds, nutrients, and/or growth conditions on a plurality of microbial species selected as relevant to a presence or absence of a plurality of cosmetic or cosmetic-like conditions likely to be present in or associated with human skin site locations. As would be appreciated, in vitro high throughput assays are usually conducted using a microtiter plate: a plate containing a grid with a large number of small divots called “wells.” The wells contain chemical and/or biological substrate (e.g., living cells or proteins). Depending on the nature of the testing, changes in microbial species metabolic activity can be detected when a chemical is added to indicate whether there is bioactivity, such as by color amounts/intensity or fluorescence differences between and among various test samples and relevant controls. High throughput microliter plates typically come in multiples of 96 wells (96, 384, or 1536), so that through the use of robotics, data processing and control software, liquid handling devices, and sensitive detection methods, and a large number of chemical compounds, nutrients, and growth conditions can be evaluated efficiently. Metabolic activity of each microbial species in relation to each tested chemical compound, nutrient, and/or growth condition can be evaluated over a period of time of interest, for example, 1 hour, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours, 48 hours, or 96 hours.

[0132] A useful example of a high throughput screening system having utility in assessing the activity of a large number of chemical compounds, nutrients, and/or growth conditions on the growth of a collection of microbial species identified as relevant to a presence or absence of a cosmetic or cosmetic-like condition associated with the surface of human skin comprises phenotype microarray system (PM) for microbial cells available from Biolog, Inc. (Hayward, CA). In the specific example of this system, each PM can contain up to 95 different chemical compounds, nutrients, and/or growth conditions of interest for testing for activity against a collection of microbial species present on one or more human skin site locations. Microarray plates can be used in which the different wells of each plate are pre-loaded with different materials of interest for testing, along with a suitable growth medium. A single microbial species can be evaluated against each of a variety of test conditions in an appropriate plate configuration. Identification of an amount of metabolic activity of the tested microbial species over a period of time using the Biolog system is based on redox technology, where cell respiration operates as a consistent signal across all microbial species types. In this regard, active cell respiration results in a reduction of a tetrazolium dye and in the formation of a strong color — for example, when using the tetrazolium dye “Biolog Redox Dye Mix D,” the color changes from transparent/colorless to purple when the dye is reduced. A colorimetric measurement of this color change thus indicates which of the tested chemical compounds, nutrients, and/or growth conditions operate to improve the metabolic activity and health of the microbial species. [0133] When appropriate, other methods of measuring an amount of growth of a microbial species in a high-throughput approach can be used, as would be understood by one of ordinary skill in art. For example, an OD600 method can be used to measure a concentration of microbial species at a beginning of a test to a test point thereof. Such OD600 measurements can be used to provide relative measures of bacterial growth, for example. Also, since many bacterial species generate volatile organic compounds during growth thereof, it can be expected that methods to evaluate a presence or absence thereof can be used for screening of potential of various chemical compounds, nutrients and/or growth conditions on the growth of bacterial species of interest. Other high throughput screening methodologies to evaluate the growth (or lack thereof) of microbial species that may have relevance to the determination of a beneficial modification of growth for a collection of microbial species that may directly or indirectly influence a presence or absence of a cosmetic or cosmetic-like condition associated with a human skin site location are contemplated for use in the screening of various chemical compounds, nutrients, and/or growth conditions for use in personal care formulations as discussed further herein.

[0134] To improve the predictive value of results obtained from the high throughput screening, artificial intelligence (Al) techniques, for example machine-learning (“ML”) and deep-leaming (“DL”) techniques, can be employed. As would be appreciated ML/DL models can have utility in identifying interesting underlying relationships and patterns between various test results and in making predictions of activity of chemical compounds, nutrients, and/or growth conditions on microbial species having relevance to a cosmetic or cosmeticlike condition at a specific skin site location of interest. There are three types of deep-learning models: 1) supervised learning models trained using input features with a corresponding target output to approximate and find underlying non-linear relationship, and these types of models are useful in regression and prediction; 2) unsupervised learning trained only on input features to make clusters or groups among the input features; and 3) reinforcement learning trained based on rewards and penalties.

[0135] In conjunction with the various requirements associated with ingredients useful for personal care formulations, to improve predictability of the performance of such ingredients, the inventors herein have initially identified supervised learning methods as relevant to the analysis of data derived from the high throughput screening herein. In this regard, human expert review of the initial high throughput screening results has been found to enhance the value of the screening results. A human expert can analyze a report of results to confirm or validate the salience of the results for subsequent use in the methodology herein to generate an ingredient mixture having effectiveness in affecting the growth of bacterial species present in or associated with a skin site location of interest.

[0136] After generation and analysis of the results of the first high throughput screening procedure, a second high throughput screening can be conducted to identify chemical compounds, nutrients, and/or growth conditions that not only are likely to affect the growth of one or more microbial species present in a skin site location that are relevant to a cosmetic or cosmetic-like condition of interest thereon, but also to generate information that can be used to provide an associated cosmetic or cosmetic-like modification to a skin site location in a person in need thereof.

[0137] In this second high throughput screening step, chemical compounds and growth conditions can be selected for further testing. Such selected compounds and conditions can be identified as actually or potentially affecting the growth of microbial species present in or associated with a human skin location where a presence or absence of cosmetic or cosmeticlike condition may also be directly or indirectly influenced by an amount of such microbial species at that location. In this regard, ingredients found to affect the growth of beneficial and non-beneficial microbial species, such as those set out in the Tables hereinabove, can be analyzed for appropriateness for use in personal care formulations suitable for use in a skin site location. That is, while a chemical compound, nutrient, and/or growth condition may be shown in the first high throughput screening step to be effective to affect the growth of one or more relevant microbial species, such test parameters will still need to be validated to ensure that they are, in fact, appropriate for use in the context of a product intended for use on human skin or scalp areas.

[0138] In this regard, given the large number of data points returned from an initial high throughput screening step, an extensive library of chemical compounds, nutrients, and growth conditions can be generated to populate a preliminary ingredient library. Some test parameters returned from the initial screening as being effective to increase or decrease the growth of a microbial species that may be relevant to a cosmetic or cosmetic-like condition at a skin site location might natively be suitable for use in skin and scalp care applications, for example, they may be assigned INCI names that indicate appropriateness for use in such applications. Such information might be found in a database, the relevant scientific literature, and/or from the professional experience of a human expert tasked with review of the preliminary ingredient library. [0139] Other materials showing an effectiveness to influence the growth of a microbial species relevant to a cosmetic condition or cosmetic-like condition at a skin site location may comprise materials that are known to be useful as ingestible prebiotic ingredients. While the effectiveness of such ingredients will first need to be validated as effective in regards to microbial species present in or associated with the skin or scalp, if such ingredients are identified as effective in enhancing the growth of a microbial species of interest — that is, exhibits prebiotic activity in the context of a skin-relevant microbial species — the material could be of interest for incorporation in a personal care formulation intended for use in a personal care ingredient having utility for skin or scalp care products.

[0140] Some of the ingredients identified as effective to increase the growth of a microbial species in the initial high throughput screening might not be suitable for use in skin care products. For example, an ingredient returned from the initial high throughput screening as being effective to enhance the growth of a beneficial microbial species present in or associated with a skin site location might nonetheless be associated with an unpleasant odor (e.g., fish, rotten eggs, etc.). It follows that such an ingredient would likely be undesirable for use in a personal care formulation. In another example, an ingredient returned from the initial throughput screening as effective to slow the growth of a non-beneficial microbial species may nonetheless exhibit known stability problems when formulated in personal care products, such as being unstable at the pH’s at which skin care products are typically formulated (e.g., pH 5-8). In yet another example, an ingredient might have a strong effect on the growth of a beneficial microbial species, but that growth might only be at a pH that is not appropriate for use in a skin care formulation. Such test parameters can be excluded from further use by the system using training information associated therewith. A human expert can also review the results, such as by reviewing a data presentation in the form of data summaries (e.g., a dashboard, report, etc.) The human expert can select or deselect various chemical compounds, nutrients, and/or growth conditions for further examination in the second high throughput screening procedure. For large data sets, computer scripts can be written to process the data summaries and deselect compounds that display the above- mentioned undesirable features.

[0141] In some implementations, a chemical compound, nutrient, and/or growth condition that is not suitable for use in a skin care formulation, such as when an otherwise effective ingredient may be available in too limited of a supply for commercial use, can be evaluated to determine whether a more appropriate analogue or derivative of that compound may be identified. For example, if an effective chemical ingredient cannot be procured under circumstances that are relevant to commercial-scale personal care formulations, automated analysis of chemical databases could be conducted to identify alternate ingredients having the same or similar characteristics that may be more appropriate for use in the skin care applications described herein. A human expert can also provide input in the generation of chemical compounds, nutrients, and/or growth conditions for use in the secondary high throughput screening process.

[0142] This secondary high throughput screening step can he configured to evaluate a smaller set of chemical compounds, nutrients, and/or growth conditions than in the first high throughput screening step, with a goal of enriching information relevant to modification of the growth of a plurality of microbial species that may be relevant to a presence or absence of a cosmetic or cosmetic-like condition at a skin site location of interest. The secondary screening step can also incorporate ingredients that were determined in the first screening to confirm previously generated results, such as by rescreening results showing increased or decreased growth of the microbial species being screened. In further implementations, a single ingredient showing a beneficial effect in the initial screening can be identified as belonging to a class of materials (e.g., fatty acids), and a plurality of materials in that class can be selected for secondary screening. In this regard, homologues of a chemical compound indicated as effective to modify the metabolic activity of a microbial species in the initial high throughput screening can be evaluated for an effectiveness in the secondary screening. [0143] The secondary screening can be conducted using the same high throughput assay method used in the initial screening to ensure consistency of results between screenings. Results from the secondary screening step or, in some cases, additional screenings deemed to be relevant to enriching the results from the initial and secondary screenings, can be used to generate a plurality of ingredient mixtures predicted to likely beneficially affect the growth of microbial species present in or associated with a skin site location of interest where such microbial species are known to or are expected to impart a direct or indirect influence on a cosmetic or cosmetic-like condition of a human skin site location.

[0144] As with the initial screening results, Al processes can be used to analyze the secondary screening results so that chemical compounds, nutrients, and/or growth conditions — and the interactions therebetween — can be surfaced from the large amount of data generated. Human expert review of the results can also be conducted to better ensure domain knowledge relevant to personal care formulations can also be leveraged. As would be appreciated, the human expert domain knowledge can have utility in training the models used with Al, with at least a benefit of improving the results of subsequent screening designs where the growth of microbial species having relevance to skin care formulations and other relevant product types might be of interest.

[0145] From the data generated in the second high throughput screening, combinations of individual ingredients that are predicted by the Al processes to likely favor the growth of beneficial microbial species and, optionally, to inhibit the growth of non-beneficial microbial species identified as relevant to a presence or absence of a cosmetic or cosmetic-like condition at skin site location can be generated. Such mixtures of ingredients — or “blends” — that would at least favor the growth of microbial species identified as “beneficial” in the context of a cosmetic or cosmetic-like condition of interest can be determined through either or both of the systems and manual review by a human expert.

[0146] In various implementations, a mixture of ingredients can be generated by either or both of human expert review and the system can be selected for further testing based on an assessment of a prediction of a likely effect of that combination of ingredients to both enhance the growth of at least one beneficial microbial species relevant to a cosmetic or cosmetic-like condition in a skin site location of interest. Still further, the ingredients for a mixture can be selected according to a generated prediction that the mixture will substantially not promote the abundance of one or more microbial species designated as “non-beneficial” in the context of a relevant cosmetic or cosmetic-like condition. Yet further, the ingredients for use in a mixture can be selected according to a prediction of whether the ingredient mixture will reduce or moderate a relative amount of growth of at least one non-beneficial microbial species relevant to a presence or absence of a cosmetic or cosmetic-like condition of interest for modification.

[0147] A selection of a combination of ingredients for incorporation in an ingredient mixture to be further tested for activity on various microbial species can be provided by the system and/or the human expert based on predictions that the blend ingredients can be expected to likely to cause a shift in ratio of beneficial microbial species and non-beneficial microbial species present at the skin site location of interest for modification of a presence or absence of cosmetic or cosmetic-like condition thereon when a personal care formulation comprising an effective amount of that combination of ingredients is incorporated in a formulation configured for use at that location. As further discussed herein, the personal care formulation should include ingredients that do not interfere with an activity of the ingredient mixture on the relevant microbial species. Further, the personal care formulation needs to comprise organoleptic properties acceptable by consumers for use at that location.

[0148] Prior to testing the effectiveness of a plurality of the ingredient mixtures for suitability for use in a personal care formulation configured to provide a desired cosmetic or cosmeticlike modification to a skin site location, a review can be conducted to ensure suitability of the ingredient mixtures for use in a formulation. Various parameters that can be used for reducing a number of candidate ingredients for in vivo and in vitro testing of not only effectiveness to increase a ratio of beneficial microbial species to non-beneficial microbial species relevant to a presence or absence of a cosmetic or a cosmetic-like condition at a specific skin site location can be derived from libraries of information, such as sources of academic articles, personal care formulation guidance, the domain knowledge of experts, etc. [0149] For example, mixtures of ingredients including petroleum-based ingredients can be deselected for further testing if a goal for a personal care formulation is that it comprises no petroleum-based ingredients. Mixtures including ingredients identified as likely to impart stability concerns in a personal care formulation can also be removed from further testing. Also, ingredient mixtures having an availability profile making them unsuitable for commercial applications can be removed from additional testing. Preliminary analysis of likely skin irritation generated by ingredient mixtures can also be developed and used to select or deselected ingredient mixtures for further testing.

[0150] In accordance with a significant aspect of the present disclosure, the inventors herein have determined that single ingredients do not generate suitable effectiveness in generating a desired shifting of a ratio of beneficial microbial species to non-beneficial microbial species, where the ratio is determined to be relevant to a presence or absence of a cosmetic or cosmetic-like condition at a skin site location. In this regard, the inventors have determined that mixtures comprising at least two ingredients identified preferably from the two-step high throughput screening methodology wherein preferably human review and selection/deselection of ingredients as discussed above are important to confer the desired cosmetic or cosmetic-like modification to a skin site location. Without being bound by theory, the inventors hypothesize that a combination of ingredients may promote growth of the at least one beneficial microbial species to generate a change in a ratio of beneficial microbial species to non-beneficial microbial species when used by a person in need of that cosmetic modification at a skin site location. In some implementations, the ingredient mixture can also not promote, provide a reduction or moderation of an abundance of at least one non- beneficial microbial species. Yet further, and also not being bound by theory, the effectiveness of the ingredient mixtures to impart a desired cosmetic or cosmetic-like modification to a skin site location may be associated with a synergistic prebiotic interaction of the two or more ingredients in the mixture. For example, each ingredient may impart an effect to enhance the metabolic activity of the at least one beneficial microbial species rele vant to a presence or absence of a cosmetic or cosmetic-like condition at or associated with a specific skin site location. Other reasons for the effectiveness of a mixture of ingredients over a single ingredient can be contemplated. Such reasons may depend on the types and amounts of beneficial microbial species and non-beneficial microbial species present in a skin site location that may be relevant to or associated with a subject cosmetic or cosmetic-like condition, for example, where such types and amounts of microbial species can effectively be modified by a blend of ingredients that preferentially provides suitable growth- related nutrition — that is, provides prebiotic activity — to one or more beneficial microbial species but not to the non-beneficial microbial species at that location.

[0151] A plurality of ingredient mixtures identified as promising candidates for inclusion in a personal care formulation intended to impart a cosmetic or cosmetic-like modification to a human skin site location can be tested for in vitro effectiveness in relation to at least one beneficial and non-beneficial microbial species identified as relevant to presence or absence of a cosmetic or cosmetic-like condition associated with a skin site location of interest. In this regard, a skin site location can be selected, and an in vitro activity of a plurality of ingredient mixtures can be tested on at least one beneficial and non-beneficial microbial species relevant to the cosmetic or cosmetic-like condition at that skin site can be evaluated.

[0152] In determining a preliminary effectiveness of each candidate mixture in testing of ingredients in testing in vitro and in vivo protocols, an amount of each mixture can be chosen to approximate an effective amount that would be relevant as an active ingredient in a personal care formulation. High levels of ingredient mixtures as needed to generate a desired cosmetic or cosmetic-like modification to a skin site location may cause formulation difficulties. This, in turn, could preclude use of acceptable formulation types which, in turn, could reduce consumer acceptance irrespective of the effectiveness of an ingredient mixture. [0153] As an example of the testing of candidate ingredient mixtures herein, in vitro testing of an effectiveness on an ingredient mixture in providing a cosmetic or cosmetic-like modification to a human axillary region can be conducted, where the in vitro testing methodology is configured to detect a change in a ratio of beneficial to non-beneficial microbial species from a baseline amount after incubation of the bacterial mock community. Mock bacterial communities comprising additional axillary beneficial microbial species and non-beneficial microbial species as can be implicated in generating axillary malodor can also be generated for in vitro testing of activity of candidate ingredient mixtures in effecting a change in a ratio of one or more beneficial microbial species to one or more non-beneficial microbial species. In assessing the effectiveness of a candidate ingredient mixture on modifying a ratio of beneficial microbial species to non-beneficial microbial species in a mock axially bacterial species community, a difference in the group of bacterial species designated as “beneficial” to the group denoted “non-beneficial” in relation to a presence or absence of axillary malodor as the cosmetic or cosmetic-like condition can be determined from a baseline or control to a test point.

[0154] Such in vitro testing can provide an initial measure of a change in the ratio of the at least one beneficial microbial species to the at least one non-beneficial microbial species present in the axillary mock bacterial community from a baseline— or control — level to a second ratio, where the second ratio is determined after incubation of the test sample for a sufficient period of time at a temperature reasonably approximating human use conditions. For example, the test solutions containing simulated human sweat, an amount of mock axillary bacterial community, and a test amount of a candidate ingredient mixture can be incubated for about 2, 4, 6, 8, 10, 12, 14, 16, 18, 24 or 36 hours at a temperature that approximates the axillary region in a real person — for example, about 32-40 °C.

[0155] The in vitro effectiveness of a candidate ingredient mixture in generating a change in a baseline ratio of at least one beneficial microbial species and at least one non-beneficial microbial species in an axillary mock bacterial community can be determined by assessment of an amount of metabolic activity for each bacterial species after incubation of the simulated human sweat, candidate ingredient mixture, and the mock community for a period of time and at a temperature that would represent a human use case scenario, as described previously. As detailed in the Examples hereinafter metabolic activity of the bacterial species in an axillary mock community can be used to derive an effectiveness of a candidate ingredient mixture to increase a ratio of the at least one beneficial microbial species to the at least one non-beneficial microbial species. This information can, in turn, have utility in providing information about whether one or more candidate ingredient mixtures should be subjected to further testing directed toward incorporation of that candidate mixture in personal care formulations configured for modification of a cosmetic or cosmetic-like condition in the axillary region, namely a reduction in axillary malodor in a person in need thereof.

[0156] In this regard, and as described in the Examples hereinafter, in vitro effectiveness of a candidate ingredient mixture can be determined by comparison of metabolic activity of an amount of a candidate ingredient mixture present in a test sample comprising an amount of simulated human sweat, and a mock axillary bacterial community comprising an amount to at least one beneficial microbial species as Staphylococcus sp. and at least one non-beneficial microbial species as Corynebacterium sp. as a mock axillary bacterial community. As discussed previously, an amount of metabolic activity can be determined by incorporation of a redox dye in each test sample.

[0157] In a further useful in vitro test protocol to assess axillary bacterial beneficial microbial species to non-beneficial microbial species ratio changes, an amount of volatile organic compounds generated from incubation of each candidate ingredient mixture in the presence of the simulated human sweat and the axillary mock bacterial community can be conducted. After incubation of each sample, a GC/MS analysis of a presence or absence of volatile organic compounds known to be associated with axillary malodor can be determined.

Volatile organic compounds not typically associated with presence of axillary malodor in a human can also be determined. In some implementations, a candidate ingredient mixture can be selected for further testing when an amount of volatile organic compounds typically associated with axillary malodor is reduced as compared to a control where no candidate ingredient mixture is included. Such difference in a presence of volatile organic compounds that are associated with axillary malodors as between a test sample of a candidate ingredient mixture and the control where no ingredient mixture was included can provide information indicating that the candidate ingredient mixture may have effectiveness in reducing a baseline amount of axillary malodor in a person who uses a personal care formulation configured for uses to the axillary region where an effective amount of an ingredient mixture can be delivered thereto.

[0158] Different amounts of beneficial microbial species and non-beneficial microbial species can be incorporated in an axillary mock bacterial community to simulate human axillary region conditions that may be indicative of a greater propensity to generate axillary malodor. Such differences in axillary malodor generation could be due to sex, genetic features, environmental influences, etc. Relative amounts of axillary malodor generated from a mock axillary bacterial community having different levels of beneficial and non-beneficial bacterial species can be assessed by analysis of volatile organic compounds generated therefrom. Metabolic activity differences can also be determined between different axillary mock bacterial communities. In some implementations, a candidate mixture can be selected for use in a personal care product formulated for a specific consumer’ s propensity to generate higher or lower levels of malodor, where the malodor generation directly or indirectly results from a baseline excess non-beneficial microbial species amounts relative to beneficial microbial species in that person. In other words, an ingredient mixture type suitable for use in reducing an amount of axillary malodor in a person in need thereof can be selected according to a baseline amount of non-beneficial bacterial species natively present in that person.

[0159] In one aspect, application of a personal care formulation to a human axillary region by a person in need of modification of an amount of axillary malodor from a baseline amount can result in reduce an amount of thiol-containing compounds after the person uses the formulation for at least about 3, or 5, or 7 days. In further implementations, an amount of thiol-containing compounds can be reduced at least about 25, 30, 40 or 50 % or more as measured from a baseline level when the person wears a personal care formulation comprising an effective amount of an ingredient mixture for at least about 3, or 5, or 7 days. Such reduction can be seen as long as the person uses the personal care formulation regularly, for example, daily. In some implementations, an amount one or more of 2-nonanol, 2- nonenal, 3M2H and hexanoic acid can be reduced at least about 25, 30, 40, or 50 % or more as measured from a baseline level when the person wears a personal care formulation comprising an effective amount of an ingredient mixture for at least about 3, or 5, or 7 days. In further implementations, an amount of one or more of the volatile organic compounds listed FIGS. 6B and 7 can be reduced by at least about 25% from a baseline level when after the person uses the formulation for at least about 3, or 5, or 7 days.

[0160] As a surprising finding, the ingredient mixtures have been shown to generate a change in not just an amount of volatile organic compounds in a human axillary region, but also the type of volatile organic compounds. In this regard, the ingredient mixtures herein can generate an increase in an amount of volatile organic compounds that are not typically associated with axillary malodor, that is thiol-containing materials. As shown in Tables 5 and 6 in the Examples hereinafter, changes can be observed in the types of volatile organic compounds resulting from use of the ingredient mixture denoted “Blend 83.” It can be expected that other volatile organic compounds that are not normally associated with axillary malodor might also be modified with various other ingredient mixtures that can be derived from the methodology herein. The volatile organic compounds identified from Blend 83- containing hybrid cosmetic sticks are set out in the Examples and accompanying Figures hereinafter.

[0161] Following or in conjunction with in vitro testing of candidate ingredient mixtures, in vivo testing of an effect of a mixture on a presence or absence of axillary malodor in the person can be conducted. In this regard, in vivo testing of an effectiveness of a candidate ingredient mixture in modifying a ratio of at least one axillary beneficial microbial species to at least one axillary non-beneficial microbial species can be conducted.

[0162] For example, swabs of each axillary region of a plurality of human volunteers can be obtained as a baseline measurement of an amount of at least one beneficial microbial species and at least one non-beneficial microbial species in each person. For this in vivo testing, a commercially relevant amount of each candidate mixture can be applied to a first axillary region of a plurality in the human volunteers, and a control of no candidate mixture in the person’s other axillary region can be provided. As shown in the Examples, swabs of the human volunteer’s axillary regions can be obtained after a period of use of a candidate mixture, and an amount of volatile organic compounds after application of the mixture or a personal care formulation including the mixture can be determined. If a reduction in an amount of volatile organic compounds after application of a commercially appropriate amount of a candidate ingredient mixture can be determined in relation to the baseline and control swabs after incubation of axillary material obtained from swabbing of the human volunteers, information associated with an effectiveness of that candidate mixture to reduce a baseline amount of axillary malodor in each volunteer can be derived.

[0163] In another in vivo testing example, swabbed axillary material can be cultured on plates that are configured to be selective for beneficial microbial species and non-beneficial microbial species relevant to an axillary region. Differences in an amount of the growth of each microbial species as compared to controls can be determined to generate information associated with an effectiveness of a candidate mixture to modify a ratio of at least one beneficial microbial species to at least one non-beneficial microbial species in a human axillary region.

[0164] The acceptability of a personal care formulation incorporating an effective amount of a candidate ingredient mixture can be determined by obtaining assessments from human volunteers of whether and to what extent they found the organoleptic properties of the formulation acceptable for use. For example, the information about how each volunteers views the spreadability, feel, and smell of a personal care formulation including a candidate ingredient mixture upon application thereof can be generated.

[0165] Referring the scalp area as a skin site location of interest for modification of a ratio of at least one beneficial microbial species to at least one non-beneficial microbial species in order to affect a cosmetic modification so as to impart a cosmetic benefit, candidate ingredient mixtures can be identified from the high throughput screening as discussed above. A new mock “dandruff” bacterial species community as discussed hereinabove and as described in the Examples can be used. In vivo and in vitro effectiveness of the candidate ingredient mixtures can be determined, and the results thereof used to identify ingredient mixtures for testing in personal care products configured to deliver the cosmetic modification associated with a change in a ratio of the at least one beneficial microbial species and the at least one non-beneficial microbial species in the scalp as a skin site location.

[0166] As shown in the Examples and accompanying Figures hereinafter, the ingredient mixtures have utility in reducing an amount of peroxidation of squalene. This is a highly relevant finding with regard to an attendant cosmetic or cosmetic-like modification associated with redness, irritation, dryness, and/or flaking in a human scalp. In this regard, squalene, a key sebum component, undergoes increased peroxidation in dandruff- affected scalps. This heightened peroxidation typically results in significantly elevated ratios of squalene monohydroperoxide (SQOOH) to squalene. Notably, the yeast Malassezia, prevalent on human scalp regions, can play a crucial role in this process. Malassezia contributes to an increased production of squalene monohydroperoxide, potentially exacerbating the oxidative stress on the scalp which, in turn, can be associated with an undesirable cosmetic or cosmetic-like condition in the scalp that can present as redness, irritation, dryness, and/or flaking. As shown in the Examples and accompanying Figures, as compared to a baseline measurement after a washout period, a shift was seen as reduced abundance of S. capitis and an attendant increase in an abundance of S. epidermidis after use of an ingredient mixture selected for use on the scalp. A reduction in the abundance of Malassezia species was observed for all participants compared to both baseline and after washout. From the baseline values, it can be understood that most of the human volunteers were at least partially symptomatic of scalp-related health issues after they stopped use of haircare products for 5 days. It follows that the shift in beneficial microbial species to non-beneficial microbial species from use of a shampoo formulation including an effective amount of the ingredient mixture generated a cosmetic or cosmetic-like modification in these person’ s scalp region, namely, a reduction in an amount of redness, irritation, dryness, and/or flaking. The sample photographs appear to confirm these results.

[0167] As with the axillary testing, candidate ingredient mixtures should be tested with human volunteers to determine an acceptability of the formulation. In the context of a scalp treatment, the formulation can be formulated as a rinse off treatment, for example, as a shampoo. As would be appreciated, active ingredients intended to improve a cosmetic condition of hair and scalp are typically delivered in shampoo form, and this type of formulation would thus he expected to be accepted by consumers for delivery of the ingredient mixtures of the present disclosure. Alternatively, a leave in serum or scalp product can be used. Evaluation of the acceptability of an ingredient mixture in a hair care formulation should also consider an effect of the formulation on the feel of the hair, as it will be difficult to convince a person to use a formulation if the hair is left in an unacceptable cosmetic condition, even when the ingredient formulation provides excellent improvement to the scalp.

[0168] Human volunteer opinions about an effectiveness of personal care formulations including an amount of ingredient mixture intended to generate a cosmetic or cosmetic-like modification to a skin site location can also be obtained. For example, human volunteers can be asked to provide personal assessments of how well a personal care formulation worked on their body sites. Human volunteers can also be assessed by others about an effectiveness of personal care formulations. For example, for personal care formulations including ingredient mixtures intended to change an amount of axillary malodor, human volunteers can conduct a self-assessment of whether an amount of axillary malodor is different and/or more or less acceptable after the personal care formulations including the ingredient mixture has been used for a period of time. Third party opinions of an amount and/or acceptability of axillary malodor in another person can also be generated.

[0169] A personal care formulation should, of course, include an amount of an ingredient mixture that is sufficient to deliver a desired prebiotic activity to the at least one beneficial microbial species present in a skin site location. In various examples, the amount of an ingredient mixture included in a personal care formulation can comprise from about 0.5% to about 5% in the personal care formulation. In some implementations, an amount of ingredient mixture relevant for use in a personal care formulation can be from about 0.1 to about 5% by weight of the formulation. Yet further, an amount of ingredient mixture having relevance to a commercially viable personal care formulation can comprise from about 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 3, 3.5, 4.0, 4.5 and 5.0 % (w/w), where any value can form an upper or lower endpoint as appropriate.

[0170] A sample formulation having effectiveness in delivering an ingredient mixture denoted “Blend 83” in the Examples hereinafter is illustrated in Example 1 as a “Hybrid Cosmetic Stick.” The ingredient mixtures are water soluble and, as such, should be dispersed in water for delivery in a water-phase during formulation. Other suitable formulations for axillary personal care products, as well as other types of formulations suitable for various skin site locations, can be determined by one of ordinary skill in the art without undue experimentation. For example, the effectiveness of an ingredient mixture as delivered from a suitable personal care formulation both upon formulation and after storage thereof under commercially relevant conditions can be determined by one or more of the tests set out in the Examples hereinafter.

[0171] Yet further, an amount of an ingredient mixture included in a personal care formulation configured for imparting of a cosmetic or cosmetic-like modification that is a reduction in a person’ s baseline level of axillary malodor can differ between persons who have differing levels of non-beneficial microbial species natively present in their axillary regions. Differing needs in humans as related to their baseline levels of axillary malodor as related to an amount of non-beneficial bacterial species natively present therein can be accommodated by different amounts of ingredient mixtures included in personal care formulations. For example, personal care formulations can be labeled as “regular” and “extrastrength” in relation to greater amounts of an ingredient mixture in the later formulation.

[0172] As noted, in order to have utility for use in a personal care formulation, the prebiotic activity of a candidate ingredient mixture must be deliverable in an amount effective to generate a change in the ratio of the beneficial microbial species to the non-beneficial microbial species natively present in a person’s skin site location. This means that the ingredients of a personal care formulation should not negatively affect the ability of the ingredient candidates to modify a ratio of at least one beneficial microbial species and at least one non-beneficial microbial species relevant to a cosmetic or cosmetic-like condition of interest for modification at a skin site location The percentage of prebiotic activity can be determined as compared to a prebiotic activity of the same mixture in relation to the same at least one microbial species when not delivered from the personal care formulation. An effectiveness of a personal care formulation to convey the intended activity on at least one beneficial microbial species can be determined, as set out in the Examples hereinafter. [0173] Moreover, the inventors have determined that an amount of prebiotic activity generated by some ingredient mixtures can be depleted by certain ingredients commonly used in personal care formulations. For example, some shampoo formulations can interfere with delivery of prebiotic activity as compared with neat solutions of the same ingredient mixture. Accordingly, an aspect of the disclosure herein comprises confirming that ingredients selected as components of personal care formulations in which the ingredient mixtures are incorporated for delivery of a prebiotic effect to one or more beneficial microbial species natively present in the skin site location of a real-life person substantially do not interfere with the intended delivery of the prebiotic activity to the beneficial microbial species of a person’s skin site location in actual use conditions. In this regard, ingredients for use in a personal care formulation including an ingredient mixture selected to delivery prebiotic activity to one or more beneficial microbial species in a person’s skin site location can be selected so that at least a majority of the prebiotic activity is, in fact, delivered to the skin site location in actual use conditions. In some implementations, greater than about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90% or 95% of an amount of prebiotic activity associated with the ingredient mixture as relevant to at least one beneficial microbial species at a skin site location can be delivered from a personal care formulation. Determination of an amount of prebiotic activity delivery from personal care formulations including an ingredient mixture intended to deliver such activity to a person’s skin site location can be determined using one or more methods set out in the Examples herein.

[0174] Further, and as would be appreciated by one of ordinary skill in the art, some of the prebiotic activity may be reduced when an ingredient mixture is incorporated in a personal care formulation configured for a body site location and that formulation is stored for later use by a consumer. However, each personal care formulation should be subjected to appropriate stability testing to ensure that relevant prebiotic activity is preserved in the formulation under typical storage and use case scenarios relevant to a particular formulation. [0175] Notably, however, preservatives that are commonly used in personal care products might interfere with a prebiotic effect intended to be conveyed to the skin site location. In this regard, preservatives used in a personal care formulation can exhibit an anti-microbial effect on both beneficial and non-beneficial microbial species natively present in a person’s skin site location because preservatives selected to eliminate microbial growth in a formulation may also act on the native microbial species. Such anti-microbial activity generated from preservatives used in the formulation can be unintended and/or undesirable at least because any change in a ratio of beneficial microbial species to beneficial microbial species resulting from use of a personal care formulation at a skin site location would not be imparted solely by the ingredient mixture, but rather by both the ingredient mixture and any preservatives in the formulation if the preservatives can suitably act on the subject microbial species. Such incidental activity on the beneficial microbial species and non-beneficial microbial species provided by the preservatives would be expected to modify the amount microbial species natively present at a person’s skin site location in a manner that would be different from prebiotic activity delivered from the ingredient mixture. To this end, a beneficial ratio change in beneficial microbial species to non-beneficial microbial species in a skin site location may not be provided when a personal care product is used by a consumer if the preservatives used therein exhibit anti -prebiotic activity on the beneficial and/or non-beneficial microbial species as relevant to a skin site location and an associated cosmetic or cosmetic-like condition at that skin site.

[0176] An aspect of the present disclosure includes selection of preservative materials that substantially do not exhibit any anti-microbial activity on any of the microbial species for which an ingredient mixture is intended to provide relevant activity. In this regard, preservatives selected for use in the personal care formulations in which ingredient mixtures are incorporated for delivery of prebiotic activity to a skin site location substantially do not affect a growth of either of the beneficial microbial species or non-beneficial microbial species present at the skin site location. Still further, any effect on the abundance of a beneficial microbial species for which an ingredient mixture is included in a personal care formulation is provided substantially by the ingredient mixture and not by any ingredients therein, including preservatives.

[0177] Appropriate preservatives that can be effective to suitably prevent a personal care formulation from microbial contamination in use while also not substantially effecting the growth of beneficial microbial species and — in some aspects, non-beneficial microbial species — as present on the skin site location, can be determined by one of ordinary skill in the art. For axillary personal care product formulations, the inventors have found the following preservatives can be useful: phenoxyethanol, 1 ,2-hexanediol, and antimicrobial peptides derived from Lactobacillus sp.

[0178] To enhance an ability to suitably preserve personal care formulations configured to deliver prebiotic activity to one or more beneficial microbial species on a skin site location, it can be beneficial to use formulation types that may need less preservation in the first order. A reduction in an amount of water in a formulation can reduce a need for preservatives. A formulation type from which the activity of an ingredient mixture can be delivered from a formulated product for a period of time that meets a minimum stability criteria for a personal care formulation, such as about 3, 6, 9, 12, 18, 24, or 36 months after manufacture thereof. The pH of a water-containing formulation can be kept as low as practicable because microbial growth during storage can be increased at higher pH’s. In this regard, the pH of the personal care formulation can be from about 3 to about 7.0 or less.

[0179] Some types of packaging can reduce a need for preservatives in a personal care formulation. For example, airless pump containers can reduce air exposure, preventing contamination and oxidation. Vacuum-sealed packaging also helps extend product shelf life by minimizing contact with external contaminants. Light-blocking packaging, such as opaque or tinted bottles, can protect light-sensitive ingredients from degradation. Single use packaging can sometimes be appropriate for storage and delivery of a personal care formulation.

[0180] Fragrances may be relevant to commercially acceptable personal care formulations that incorporate the ingredient mixtures as relevant to the intended cosmetic modifications on a skin site location. Concentrations of fragrances may impact whether or not they are appropriate for use in personal care formulations intended to impart a cosmetic or cosmeticlike modification in a human skin site location. Fragrances that exhibit bactericidal effects at some concentrations, such as many essential oils, may be contraindicated for the same reasons that certain preservatives may not be appropriate for use.

[0181] While Examples of beneficial microbial species and non-beneficial microbial species in the human axillary region and the scalp areas are described hereinafter, it is to be understood that the information derived from the high throughput screening herein can have utility in generating personal care formulations that can confer cosmetic or cosmetic-like modifications to other skin site locations. Pertinent in vitro and in vivo testing can be conducted to identify one or more candidate ingredient mixtures for use in a personal care formulations intended to impart a cosmetic or cosmetic-like modification in a human skin site location. As set out above in Table 4 hereinabove, different skin site locations are associated with different beneficial microbial species and non-beneficial microbial species. It follows that collections of microbial species communities can be created for any skin site location where a change in a ratio of at least one beneficial microbial species to at least one non- beneficial microbial species can have relevance to a generation of a desired cosmetic or cosmetic-like modification therein. Top candidate blends generated from the high throughput screening can be selected for in vitro and in vitro testing. Specific tests can be designed to determine an effectiveness of the candidate ingredient mixtures to impart a change in a ratio of at least one beneficial microbial species to a non-beneficial microbial species in a subject location. Delivery of the activity ingredients — that is, the candidate ingredient mixtures — can be assessed to ensure that a personal care product in which the ingredient mixture is included will impart the intended cosmetic or cosmetic-like modification to a skin site location in need thereof.

EXAMPLES

[0182] The following Examples illustrate aspects of the heretofore described methodology to modify a ratio of beneficial microbial species to non-beneficial microbial species as relevant to imparting a cosmetic or cosmetic-like modification to a human skin site location in need thereof.

EXAMPLE 1: Sample Ingredient Mixture Candidates Identified as having Potential for Prebiotic Activity Relevant to Shift the Amounts of Beneficial Microbial Species to Non- Beneficial Microbial Species in a Human Axillary Region

[0183] Selection of Ingredient Mixtures for In Vivo and In Vitro Testing: The Examples were derived a multi-stage high throughput screening procedure as described above. Ingredient mixtures identified by the high throughput testing were reviewed by a human expert for confirmation that a system-identified ingredient mixture was appropriate for further testing. For example, ingredients that were not likely to meet formulation guidance parameters due to availability, ingredient amounts needed for necessary activity, organoleptic properties, etc. were excluded from in vitro and vivo testing. It should be appreciated that the system-generated data provided suggested ingredient mixtures predicted by individual results from the high throughput testing. That system-generated data was then evaluated by a human expert prior to testing thereof as set out below.

[0184] As set out hereinbelow, the in vivo and in vitro testing of the ingredient mixtures was conducted to validate the predicted efficacy of the generated ingredient mixtures to modify a starting ratio of beneficial microbial species to non-beneficial microbial species as relevant to a cosmetic or cosmetic-like modification in vitro and in vivo testing scenarios, including incorporation of some ingredient mixtures in personal care formulations and testing thereof. Activity of human selected ingredient mixtures was evaluated to determine the effectiveness to modify — or shift — a ratio of beneficial microbial species to beneficial microbial species relevant to a cosmetic or cosmetic-like modification in a subject skin site location — namely, human axillary and scalp regions — and the data generated therefrom was used to select ingredient mixtures for further testing in a suitable personal care formulation.

[0185] It should be noted that only a subset of the total collection of ingredient mixtures identified by the system were selected for initial validation of the predicted activity. It follows that many of the system-generated ingredient mixtures may exhibit relevant activity on various bacterial species that may be beneficial to a skin site location. These ingredient mixtures that can be later determined to exhibit relevant activity to shift a ratio of beneficial microbial species to non-beneficial microbial species in the axillary region (or any other region of interest) can also have relevance for use in personal care products.

[0186] Mock Axillary Bacterial Species Community: A Mock Axillary Bacterial Species Community was generated for screening of the ingredient mixtures selected for further testing of prebiotic activity that could be relevant to use thereof in a personal care product intended for use in a human axillary region.

[0187] A co-culture plate was set up with each experimental well containing a total volume of 1500 pL consisting of 150 pL of blend/diluent control, 850 pL of SLM(+TSB) and a mock community having 100 pL each of .S', epidermidis, S. hominis, C. acnes, C. jeikeium, and C. tuberculostearicum precultures that had been normalized to 5xlO 7 CFU/mL. The mock bacterial species community had the following makeup: C. jeikeium (20%), C. tuberculostearicum (20%), S. epidermidis (20%), .S’, hominis (20%) , C. acnes (20%), meaning that the initial quantity of Staphylococcus sp. was 40% and the initial amount of Corynebacterium sp. was 80%. In order to observe a meaningful change in an initial ratio of Staphylococcus sp. to Corynebacterium sp.— as the beneficial axillary microbial species and non-beneficial microbial species, respectively— from incubation of a mock bacterial species community having relevance to an axillary region, an excess of Corynebacterium sp. to Staphylococcus sp. was used.

[0188] For a candidate ingredient mixture to be assessed as promising for use, a goal was to modify a ratio of the bacterial species in the test material from an initial % of Staphylococcus sp. and Corynebacterium sp. In other words, the desired result for the initial in vitro testing of ingredient mixtures generated from the high throughput screening process was to determine whether the activity predicted by the machine learning model was experimentally observed. [0189] Simulated Human Sweat: A simulated human sweat formulation, denoted “sweatlike media” (“SLM”), was prepared for the in vitro testing of the effects of various ingredient mixtures was prepared using a variation of the methodology described in Oates, Angela, and Andrew J. McBain. "Growth of MRS A and Pseudomonas aeruginosa in a fine-celled foam model containing sessile commensal skin bacteria." Biofouling 32.1 (2016): 25-33.

[0190] As used in the in vitro experimental procedures hereinafter, the SLM was prepared as follows.

[0191] Table 5: Sweat- Like Media Preparation

[0192] An amount of each ingredient mixture was incorporated in the SLM at an effective amount approximating an in vivo use case in a personal care formulation of 0.1 % to prepare a test solution for in vitro screening purposes.

[0193] Mean difference calculations in relation to the shifting of the ratio of beneficial microbial species to non-beneficial microbial species as relevant to a cosmetic or cosmeticlike condition — here an amount of axillary malodor — in a test solution over the course of an in vitro test process as shown by the abundance of each bacterial species was determined using a screening process whereby each ingredient mixture was incubated over a 48-hour period. DNA was extracted at timepoints Oh, 17h, 24h, 41h and 48h distributed across the 48- hour period, then qPCR was performed using species-specific probes using the DNA extracts as template. Mean differences were calculated based on Ct values generated by qPCR. For an ingredient mixture to be assessed as promising for use, a goal was to modify ratio of the bacterial species in the test material from an initial % of Staphylococcus sp. and Corynebacterium sp. in the Mock Axillary Bacterial Species Community at the start of an incubation step to a ratio where there was more Staphylococcus sp. present in the test solution by the end of the incubation process.

[0194] The following ingredient mixtures identified by the high throughput method were selected by the human expert for testing.

[0195] TABLE 6: Ingredient Mixtures Selected for In vitro Screening

[0196] Mean difference calculations from a starting point at baseline and after incubation for ingredient mixtures selected for further screening are set out in Table 7 below. [0197] Table 7: Mean Difference Calculations after In vitro Screening of Various Ingredient Mixtures

[0198] Blend 83 and Blend 50 were initially selected by a human expert for further testing. Later tests incorporated a comparison of Blend 83 and Blend 2 as set out hereinafter. Such selection was based not only on the mean difference value, but also evaluation of information such as ingredient cost, stability in a cosmetic formulation, availability, cosmetic appropriateness, etc. Ingredient mixtures that were generated by the system but not screened in these Examples, could still be useful for use in personal care formulations configured to impart a cosmetic or cosmetic-like modification to a human axillary region, as would be appreciated.

EXAMPLE 2: Testing of Blend 83 and Blend 50 for In Vitro Effect on Different Bacterial Species Appearing in a Human Axillary Region

[0199] Blend 83 and Blend 50 were incorporated in a sample personal care formulation — namely, a Hybrid Cosmetic Stick — to determine an activity in vitro of these ingredient mixtures to impart a shift in a ratio of axillary beneficial microbial species to non-beneficial microbial species in an in vitro test.

[0200] Table 8: Hybrid Cosmetic Stick

[0201] Procedure to make Hybrid Cosmetic Sticks: Dissolve ingredient mixture in water. While mixing, add Glyceryl Stearate (and) Polyglyceryl-6 Palmitate/Succinate (and) Cetearyl Alcohol and heat to 75°C. Then separately combine Phase B ingredients and heat to 80°C. Begin cooling, then add Phase A into Phase B at 76-78°C and mix well. Slowly incorporate Phase C and mix until homogeneous. Pour into molds at 75 °C.

[0202] For comparison testing, “Placebo” or “blank” Hybrid Cosmetic Sticks were also generated using the same ingredients having the above composition, less the added ingredient mixture being tested, with the additional amount being QS as noted.

[0203] In vitro testing procedure for Blends 83 and 50: In order to collect additional knowledge about a specificity, or lack the thereof, of Blend 83 and Blend 50 on the individual bacterial species implicated and not implicated in the generation of axillary malodor, S. epidermidis was included as a beneficial (non-odorous) bacterial species and the non- beneficial microbial species were Corynebacterium sp. and S. hominis.

[0204] 2mL SLM normalized to a starting OD600 of 0.02 for each bacterial species and then each mixture was combined with 100 mg of the Anhydrous Cosmetic Stick each incorporating either Blends 83 or Blend 50 included at 4 % (w/w) to generate an effective concentration of these tested ingredient mixtures of 0.2% in the test solution.

[0205] Results: FIGS 1A and IB show the results of testing with Blend 83/Hybrid Cosmetic Stick at 6 hours. Blend 50/ Hybrid Cosmetic Stick results are shown in FIG. 1C and ID. [0206] To summarize, in a collection of microbiota indicative of bacterial species typically present in a human axillary region cultured with SLM and Blend 83 formulated in the Hybrid Cosmetic Stick at 4% (w/w), an increase in S. epidermidis growth was observed as compared to the Placebo Hybrid Cosmetic Stick. Significantly, the growth of Corynebacterium sp. and S. hominis decreased as compared to the Placebo Hybrid Cosmetic Stick, thereby indicating that those bacterial species typically associated with the presence of axillary malodor were affected by Blend 83, even while the amount of S. epidermidis remained less affected by the Blend 83 Hybrid Cosmetic Stick.

[0207] Blend 83 can therefore be hypothesized to be effective to reduce the amount of those bacterial species often implicated in producing odorous volatiles in the axillary regions of humans — that is Corynebacterium sp. and S. hominis — while having a lesser effect on a S. epidermidis. In some contexts, the ingredients in Blend 83 could be considered to exhibit a prebiotic activity on at least S. epidermidis. Put another way, Blend 83 can be shown to shift a ratio of beneficial bacterial species to non-beneficial bacterial species present in a human axillary region, where the shifting can be directionally inferred to be relevant to an appearance of axillary malodor in a human who uses a personal care formulation incorporating an effective amount of Blend 83 for a sufficient period of time.

[0208] Blend 50 demonstrated a decrease in all bacterial species present, thus showing that this composition exhibited a “broad spectrum” activity — at least in relation to the bacterial species examined. These results showed that the ingredients in Blend 50 exhibited a reduction in the bacterial species typically associated with axillary malodor, as well as those species that are not so associated. It would then appear that Blend 50 does not exhibit prebiotic effect on S. epidermidis.

[0209] Given the effectiveness of Blend 50 in killing substantially all of the bacterial species in the SLM, the inventors concluded that, while the information generated from the machine learning model and as reviewed by the human expert correctly predicted an efficacy of the materials on affecting the bacterial species normally associated with a human axillary region, Blend 50 did not provide the specific intended effect of increasing an amount of beneficial bacterial species to non-beneficial microbial species over a baseline amount because no S. epidermidis remained after 6 hours of contact with this ingredient mixture. In other words, Blend 50 did not exhibit prebiotic activity on S. epidermidis. Blend 50 also did not generate a shift in a ratio of beneficial to non-beneficial axillary bacterial species as tested in this Example.

EXAMPLE 3: Examination of Blend 83 on Cultured Human Axillary Bacterial Species

[0210] An examination of axillary swabs collected from 4 human subjects was conducted to assess the effects of Blend 83 on human axillary bacterial species typically associated with malodor, that is, “non-beneficial bacterial species” in the context of the axillary region. Swabs of the volunteer’s axillary regions were obtained, and the samples were incubated in Corynebacterium sp. and Staphylococcus sp. specific plates. Controls of axillary swabs from the volunteer corresponding axillary region in which no Blend 83 was applied were used for comparison.

[021 1] Results: These results demonstrated that Blend 83 generated a distinct decrease in Corynebacterium sp. and Staphylococcus sp. Blend 83 appeared to generate a shift in the content/amount of the bacterial species on the skin of these subjects, as opposed to a complete killing of the microbiota.

[0212] Note that this protocol cannot distinguish between 5. hominis and S. epidermis. Note also that the inventors did not measure skin response for different individuals in these experiments. However, the inventors currently understand that the specific characteristics of each person (e.g., skin pH, sweat propensity, health condition, age, location, food intake, medications etc.) may influence the resulting odor and skin bacterial species characteristics in an individual.

EXAMPLE 4: GC-MS Testing of Presence/Absence of Malodorous Compounds in Mock Axillary Bacterial Species Community

[0213] GC-MS testing was conducted to examine effects of Blends 83 and 50 on bacterial species known to be associated with the presence of malodorous compounds in the human axillary region.

[0214] A microbial community of Corynebacterium sp., S. hominis and S. epidermidis, at a starting OD600 of 0.02 for each strain, was generated as Mock Axillary Bacterial Species Community B. Inoculum volume was determined based on what would result in a normalized starting OD of 0.02 in the final culture.

[0215] With regard to Samples 1-7, filtered and sterilized real-life human sweat was examined with and without the Blends 83 and 50. The Mock Axillary Bacterial Species Community from Table 9 below, was inoculated into 1 mL synthetic SLM supplemented with 1 mL filtered and sterilized human sweat obtained from the axillary region of human volunteers. [0216] TABLE 9: Mock “Axillary” Bacterial Species Community

[0217] This human sweat was tested against Blend 83, Blend 50, and a no-blend control. The cultures were incubated at 37 °C for 24 hours. Following incubation, the cultures were analyzed by GC-MS for the liberation of 3M2H and isovaleric acid. The effective amount of each ingredient mixture in the test solution in Samples 1-7 was 0.2%.

[0218] As would be appreciated, real-life human sweat served as a natural source of biological precursors necessary for the bacteria to generate odorous volatile compounds from human axillary areas. To this end, it was hypothesized that this actual human sweat could provide naturally occurring precursors to enable the mock axillary bacterial species communities to produce odorous compounds for analysis in GC-MS testing.

[0219] To generate human sweat samples for testing associated with Samples 1-7, volunteers were asked to wear gauze pads underarms to collect sweat during daily activities. The gauze was centrifuged to extract the human sweat and the extracted sweat was passed through a 0.2 micron filter to sterilize prior to use. [0220] Instead of human-derived sweat, Sample #’s 8-18 used 3M2H-Gln as a 3M2H precursor to assess the effects of the test compounds on malodorous compounds known to be present in the human axillary region. Controls were also prepared. It was hypothesized that if Blends 83 and 50 worked to reduce or eliminate 3M2H-Gln, as shown by GC-MS chromatograms, the compositions would also exhibit effectiveness in reducing axillary malodor when used by persons in personal care formulations that included such mixtures of ingredients.

[0221] An additional sample that was a “modified” Blend 50 that incorporated an amount of yeast extract as identified by a human expert to determine whether the absence of S. epidermidis at the end of incubation in Example 2 with the previous Blend 50 could be due to an absence of prebiotic material effective to enhance the growth of this beneficial microbial species in the blend. Y east extract is known to exhibit a prebiotic effect on several bacterial species, including S. epidermidis.

[0222] Table 10: “Modified” Blend 50

[0223] Samples 8-18 that were tested using GC-MS were prepared using the following procedure: a mixed microbial community comprising Mock Axillary Bacterial Community in Table 9, at a starting OD600 of 0.02 for each strain, was inoculated into 2.5 mL synthetic SLM with lOmM of 3M2H-Gln, and tested using 4% (w/v) Blend 83, 4% (w/v) Blend 50, 4% “modified blend” 50 (w/v) (as shown in Table 4) and a no-blend control.

[0224] The experimental set up is shown in Table 11 below. [0225] Table 11: GC-MS Analysis of Malodorous Compound Development with and without Blend 83, Blend 50, and Modified Blend 50 (Controls included)

Note 1: SLM was ImL and the human sweat was ImL for a total of 2mL.

Note 2: 2.5mL SLM was added the 3M2H-Gln concentrated at a final concentration of lOmM would have 62.5pL amount based on the stock concentration.

[0226] Results: The GC-MS data showed that each of the samples that included Blend 83, Blend 50, and Modified Blend 50 exhibited a reduction in 3M2H peaks, as a biological degradation product of supplied precursor 3M2H-Gln. A reduction in isovaleric acid peaks (presumably a biological degradation product of L-leucine supplied in the SLM) was also observed. These results indicate that each of these ingredient mixtures can exhibit a beneficial reduction in malodorous compounds associated with human axillary malodor.

[0227] These results also confirmed the previous results of Example 2 that Blend 50 did not exhibit an ability to increase an amount of beneficial .S', epidermidis. In this regard, Blend 50 did not provide a desired prebiotic effect on at least one beneficial bacterial species present in a human axillary region, namely, S. epidermidis. The inventors concluded that while this mixture of ingredients would likely have effectiveness in reducing axillary malodor if used in a personal care formulation for use as a deodorant, Blend 50 nonetheless did not provide the desired effect of shifting the ratio of beneficial microbial species to non-beneficial microbial species, at least because the amount of remaining beneficial microbial species was effectively zero. Blend 50 did not provide a desired prebiotic effect on at least one beneficial bacterial species present in a human axillary region, namely, S. epidermidis.

[0228] Notably, the incorporation of yeast extract did not impart prebiotic activity to the Blend 50 ingredients. This indicates that generation of prebiotic activity requires more than addition of an ingredient that is known in the prior art to convey prebiotic activity. Instead, the inventors hypothesize that an ingredient mixture preferably requires generation of a combination of ingredients preferably predicted by the machine learning process in the first order and as validated by a human expert to have effectiveness as the combination, and not on the basis of any prebiotic or non-prebiotic activity of the individual ingredients.

EXAMPLE 5: Test of Bacterial Conversion of S-Benzyl-L-Cysteine to Benzyl Mercaptan Using Mock Axially Bacterial Community with Blend 83

[0229] A Mock Axillary Bacterial Species Community as in Table 9 above comprising selection of 50:50 beneficial microbial species and non-beneficial microbial species of different varieties — that is, bacterial species associated and not associated with axillary malodor-was prepared.

[0230] An amount of Blend 83 was added to a sample of the Mock Bacterial Species Community in SLM and bacterial conversion of a model substrate of S-Benzyl-L-Cysteine was analyzed as a proxy for analysis of the precursor Cys-Gly-3M3SH to odor molecule 3M3SH. A control of 1% NaHCC , a commonly used material in deodorant products, was also tested as a control in the SLM. Cultures were incubated at 37 °C for 6 hours.

[0231] Results: FIG. 2 shows that Blend 83 added at 0.4% to SLM and incubated reduced the amount of thiol produced versus the controls-SLM only and 1% NaHCCL. These results indicate that the ingredients in Blend 83 could be expected to decrease the amount of axillary malodor in a person who applies a composition incorporating an effective amount of a prebiotic mixture including Blend 83 in a personal care formulation configured for application to the axillary region.

EXAMPLE 6: Test of Blend 83 in Mock Axillary Bacterial Species Communities of SLM Having Different Amounts of Odor-Producing Bacteria Incubated in SLM

[0232] Two mock bacterial species communities were prepared having differing amounts of .S', epidermidis, as a non-odor producing bacterial species, and S. hominis, C. striatum, C. tuberculostearicum, and C. jeikeium as odor producing bacterial species. The amounts of each bacterial species are shown below in Tables 12 and 13. The levels were designated as “Mild Odor Producing” and “High Odor Producing” in accordance with the greater amounts of odor producing bacterial species in the latter, although each would be expected to produce some odorous volatile organic materials during incubation.

[0233] Table 12: Mild Odor Producing (“MOP”) Mock Axillary Bacterial Species Community

[0234] Table 13: High Odor Producing (“HOP”) Mock Axillary Bacterial Species Community

[0235] Each community was normalized to a starting OD600 of 0.06 and was incubated for approximately 16 hours in SLM with 10 mM 3M2H-Gln (odor precursor) with each of 0.3% and 0.4% Blend 83 (3 mL in duplicate) to approximate an amount in a personal care formulation of 3 and 4% (w/w), respectively. After incubation, 1 mL of culture was aliquoted, and DNA was extracted. 16S rRNA sequencing was then performed to determine the relative abundance of the microbes in the community. The remaining 2 mL of culture was used for GC-MS analysis. [0236] GC-MS analysis was conducted to analyze an amount of odor producing volatile compound via conversion of 3M2H-Gln (odor precursor) as a proxy for bacterial production of this odoriferous compound in a human’s axillary region.

[0237] Results: FIG. 3 shows the differences between the various levels of Blend 83 between and among the baseline amounts of .S'. epidermidis and Corynebacterium sp. in non-incubated SLM (“Start”) incorporating MOP and HOP mock microbial communities and incubated SLM (“Finish”) having MOP and HOP mock microbial communities.

[0238] The results show marked reduction in Corynebacterium sp. from addition of Blend 83 at both 0.3% and 0.4% levels in the MOP and HOP SLM samples. The improvements are also seen against NaHCOs, as a control odor reducing composition. These results could be expected to provide exceptional reduction in odoriferous compounds in those who experience high degrees of axillary malodor using the prebiotic composition disclosed herein. It follows that, in contrast to existing “extra strength” deodorants and/or antiperspirants that may reduce odor using ingredients that may be harsh or damaging to skin, Blend 83 -containing formulations would provide effective odor reduction without also causing such side effects. The odor reduction appears to be due to a shift in a ratio of beneficial microbial species to non-beneficial microbial species resulting from an activity of this ingredient mixture to provide a prebiotic effect on at least the S. epidermidis in the mock axillary bacterial species community. Moreover, as shown in Example 13 hereinafter, the ingredients of Blend 83 may even improve skin condition in regards moisturization.

[0239] While it could not be determined whether the increase in Staphylococcus sp. seen in this Example was for both S. epidermidis and .S', hominis, or only the former, given the significantly larger amounts of S. epidermidis in each of the MOP and HOP mock microbial communities at the end of incubation, any increase that might occur from the prebiotic effect of Blend 83 would still be expected to provide reduced axillary malodor when used in a topical composition intended for use in an axillary region by a person in need such cosmetic benefit.

EXAMPLE 7: Examination of “Non-Targeted” Volatile Organic Compounds Generated from Incubated MOP and HOP Samples with and without Blend 83 Addition

[0240] The inventors posited that modification of the ratio of beneficial to non-beneficial bacterial species in a human axillary region might not only be associated with a decrease in malodor, but that different volatile organic compounds may be generated when a system- generated ingredient mixture is used to modify an amount of axillary malodor. To confirm this hypothesis, as well as to ensure that unexpected and, perhaps, “unpleasant” odors could be generated along with a shift in the axillary bacterial species ratios, a GC-MS analysis of volatile organic compounds generated from incubation of volatile organic compounds generated from incubation of MOP and HOP in the presence and absence of Blend 83 was conducted. The incubated samples from Example 6 were used for the GC-MS analysis. As described previously for Example 6, each community was normalized to a starting OD600 of 0.06 in the final culture and was then incubated for approximately 16 hours in SLM with 10 mM 3M2H-Gln (odor precursor) with each of 0.3% and 0.4% Blend 83 (3 mL in duplicate) to approximate an amount in a personal care formulation of 3 and 4% (w/w), respectively. After 1 mL of each culture had been aliquoted for 16S rRNA sequencing, the remaining 2 mL culture was analyzed by GC-MS.

[0241] The volatile organic compounds examined in this Example were identified as being associated with odors that were different from those typically associated with human axillary malodor — that is, “non-targeted” axillary microbiome volatile organic compounds. All samples were normalized using matrix blanks to preclude any SLM components from the non-targeted compounds analysis. All compounds had a minimum 70 Match Factor score to the NIST database of organic compounds (https://chemdata.nist.gov/).

[0242] Results: As shown in Table 14 with the entries marked with *, certain volatile compounds often associated with “pleasant” odors were identified when Blend 83 was incubated with SLM and the axillary mock bacterial communities. Also, baseline amounts of volatile organic compounds that are often associated with unpleasant odors are reduced when Blend 83 is incubated with SLM. These results indicate that not only do the ingredients in Blend 83 reduce the presence of volatile organic compounds typically associated with human axillary malodor (i.e., “targeted volatile organic compounds”), these ingredients may also generate volatile organic compounds that can be associated with “pleasant” odors, such as flowers and citrus.

[0243] The inventors observed that most of the non-desirable odorants were produced in Corynebacterium spp. dominant (HOP) communities where no Blend 83 was incorporated. In short, these samples “smelled worse” than other samples, thus indicating that the ingredients in Blend 83 may impart additional benefits in odor properties in individuals who experience natively higher levels of axillary malodor. A qualitative review of the data from this Example is set out in the below Table. TABLE 14: Characterization of Volatile Compounds Identified via GC-MS Analysis of Incubated SLM Controls and Blend 83 Treated in SLM and Blend 83 Treated SLM in MOP and HOP In Vitro Samples

* Odor characterization and perception derived from online searches for each volatile organic compound. EXAMPLE 8: Evaluation of Effect of Blend 83 on Metabolic Activity of Axillary Bacterial Species

[0244] The effect of Blend 83 on the metabolic activity of a collection of beneficial and non- beneficial axillary bacteria was conducted.

[0245] Hybrid Cosmetic Sticks with 4% (w/w) Blend 83 were dissolved in SLM at a 10% w/v concentration. Placebo Hybrid Cosmetic Sticks having the same ingredients but without Blend 83 were dissolved in SLM at a 10% w/v concentration. The amount of Hybrid Cosmetic Stick incorporated in the SLM was selected as an estimation of an in vivo application. Metabolic activity of S. epidermidis as a non-odor producing and beneficial axillary bacterial species was compared to that of odor producing and therefore ‘non- beneficial” bacterial species that were S. hominis, C. striatum, C. tuberculostearicum, and C. jeikeium.

[0246] The metabolic activity was assessed by culturing each bacterial species normalized to a starting OD600 of 0. 1 in the 10% w/v SLM + and - Blend 83 with addition of a redox dye. In this regard, “Dye H” from Biolog is a tetrazolium salt that is reduced to a formazan product by metabolically active microorganisms. It is a redox-sensitive colorimetric dye. The reduction of the dye is coupled to the oxidation of NADH. As a result, the intensity of the redox reaction, which is directly proportional to microbial activity, can be measured spectrophotometrically.

[0247] Cultures were incubated at 37 °C and kinetic metabolic data was then collected over a 20-hour period. A sample of an incubated SLM including the mock bacterial community normalized to a starting OD600 of 0.1 and Blend 83 was treated with the redox dye and a colorimetric change that measured metabolic activity was accessed using a plate reader. The area under the curve (AUC) was calculated for each sample. A control of each of the beneficial and collection of non-beneficial bacterial species in SLM without addition of Blend 83 was also included. Each test was conducted as 3 replicates per assay, in 5 separate experimental procedures.

[0248] Results: As shown in FIGS. 4A and 4B, Hybrid Cosmetic Stick containing 4% (w/w) Blend 83 showed efficacy in reducing the metabolic activity of odor producing bacterial species S. hominis, C. striatum, C. tuberculostearicum, and C. jeikeium as measured by an in vitro metabolic assay, thus indicating an efficacy of Blend 83 for reducing axillary malodor in a human. In this regard, when incorporated at 10% (w/v) in the SLM with the bacterial species, these Blend 83 test formulations resulted in a greater than 50% reduction in the metabolic activity (as measured by AUC over the 20h culture period) of each of the malodor producing bacterial species.

[0249] Significantly, with 4% (w/w) Blend 83 Hybrid Cosmetic Stick also showed effectiveness in increasing the metabolic activity of .S', epidermidis. Compared to a placebo Hybrid Cosmetic Stick, 4% (w/w) Blend 83 hybrid sticks resulted on average in ~ 1.8 times more metabolic activity observed for non-odor producing bacterial species compared to odor producing species. This indicates that Blend 83 exhibits prebiotic activity on .S'. epidermidis under the test conditions.

[0250] These results thus confirm that incorporation of Blend 83 in the Hybrid Cosmetic Stick can be expected to modify a ratio of beneficial to non-beneficial axillary bacterial species when a personal care formulation incorporating an effective amount of this ingredient mixture used by a human in their axillary region.

EXAMPLE 9: Analysis of Bacterial Species Differences for a Group of Human Volunteers Using Genetic Sequencing

[0251] To confirm a differential activity of Blend 83 in vivo, swabs from human axillary areas were taken from 5 human volunteers were incubated using the methodology herein. An analysis of bacterial species amount was conducted at Day 0 and at Day 5 after use of the Hybrid Cosmetic Stick comprising 4% (w/w%) of Blend 83 on the right side and a Placebo Hybrid Cosmetic Stick not containing Blend 83 on the left side. The relative abundance of the bacterial community on the axillary of each participant was determined by 16S rRNA sequencing.

[0252] Results: A difference was seen in the ratios of bacterial species for each participant shown in FIG. 5A when the Hybrid Cosmetic Stick containing 4% (w/w) Blend 83 was used by each of the 5 participants in their right axillary areas. Further, as shown in FIG. 5B, there was a marked reduction in Corynebacterium sp. between Day 0 and Day 5, with every participant exhibiting a reduction in this malodor-causing non-beneficial bacterial species in their left axillary region vs. their right, with the former having been treated with the Hybrid Cosmetic Stick of Table 8 having Blend 83 therein. [0253] Table 15: Statistical analysis of differences between human axillary

Corynebacterium sp. Between day 0 and day 5

[0254] A plot of the difference in Corynebacterium sp. in these 5 human volunteers between their right and left axillary regions from Day 0 and Day 5 is shown in FIG. 5C. This plot shows that the amount of Corynebacterium sp. in the right and left axillary areas for each participant are not statistically different between Day 0 and Day 5 when compared a Placebo Hybrid Cosmetic Stick. In contrast, use of the Hybrid Cosmetic Stick including 4% (w/w) Blend 83 shows a marked decrease in the amount of Corynebacterium sp. in the participants when each used the Blend 83-containing composition for 5 days.

[0255] These results further demonstrate that use of the prebiotic Blend 83 changes — or shifts — the ratios of beneficial and non-beneficial bacterial species typically present in a human’s axillary regions when used in an anhydrous stick formulation.

EXAMPLE 10: In vivo analysis of Hybrid Cosmetic Stick with 4% (w/w) Blend 83 in Single Human Volunteer

[0256] As would be appreciated, the information generated from the machine learning protocol and human review thereof will have limited utility in generating real cosmetic improvements if there was not a beneficial effect when used by a human in actual use case scenarios. In order to confirm the effectiveness of Blend 83 when incorporated in a personal care formulation, an in vivo study was conducted. The intent of the study was to confirm that Blend 83 actually worked in humans in real-life. To this end, a detailed analysis of a change in the amounts of different types of volatile organic compounds in a human volunteer was determined.

[0257] In a first experimental procedure, a human volunteer was recruited to conduct a detailed assessment of an effect of Blend 83 on bacterial species actually present in their axillary region.

[0258] In this human volunteer study, as well as the multiple participant test in Example 1 1 hereafter, there was a washout period that lasted 12 days, with for the first 10 days the participant did not use deodorant or other underarm product and using exclusively a body wash followed by 2 days without using deodorant, other underarm products, or the provided commercial body wash (Native Unscented Body Wash, Native Company, San Francisco, CA). [0259] To obtain a sample of bacterial species natively present in a human volunteer after the washout period, a wooden cotton-tipped swab was used to sample each of the person’ s left and right axillary regions. Prior to swabbing, the swab was thermally cleaned by heating to 90 °C to destroy any baseline bacterial species that may have been present on the swab natively. The sufficiency of this thermal cleaning method was confirmed by determining that no bacterial species were present on the swab after heating.

[0260] After swabs of each of the left and right axillary regions of the human volunteer were taken as baseline measurements for volatile organic compounds derived from the person’ s natively present bacterial species, the subject applied a hybrid stick formulation comprising 4% w/w Blend 83 in their left axillary region and a Placebo Hybrid Cosmetic Stick in their right axillary region each day.

[0261] The amounts of volatile organic compounds generated from swabs of the human volunteer’s axillary regions taken before and after application of the hybrid stick formulation comprising 4% (w/w) of Blend 83 Hybrid Cosmetic Stick to one armpit but not the other were compared to the amounts and types of volatile organic compounds generated from the incubation of each sample in SLM. For each subject pair axillary region pair (L/R), a sample was obtained via a thermally cleaned wooden tipped swab. An amount of each volatile organic compound present in an incubated sample was determined via GC-MS at baseline and at the end of the study. For each subject’s armpit, the change in generated volatile organic compound amount was determined. The mean of the differences between right and left armpit for all subjects was determined. If the mean differences were less than 0, a reduction in a volatile organic compound in the right armpit relative to the left, thus indicating a likely reduction of axillary malodor in the subject, as well as an attendant change in the ratios of beneficial non-odor causing bacterial species and non-beneficial odor-causing bacterial species.

[0262] For each volatile compound observed from GC/MS analysis, the mean of the baseline amounts were determined to obtain the reference value for percentage change calculations. [0263] Calculations of differences between baseline samples and samples after use of a hybrid stick formulation containing 4% (w/w) of Blend 83 were calculated according to the following formula:

Mean difference %(Right - Left)=

Mean of [Right(End-Start)- Left (End-Start) //Mean Baseline amount * 100%

[0264] Results: There was a significant difference in the amounts of volatile organic compounds present in the human volunteer’ s left and right axillary regions after use of the 4% (w/w) Blend 83 Hybrid Cosmetic Stick. FIG. 6A shows the actual differences as determined by GC/MS analysis. FIG. 6B shows differences in mean amounts of each volatile organic compound as determined by the formula set out above for those compounds where a decrease of 25% or greater was determined. From these results, a conclusion was drawn that not only was an amount of malodor in the left axillary area reduced in the subject by use of the 4% (w/w) Blend 83 Hybrid Cosmetic Stick, but also that the formulation caused a shift in an ratio of beneficial to non-beneficial bacterial species in the subject’s left axillary area relative to a baseline — or “native” — ratio of axillary bacterial species in the subject.

[0265] This Example further confirmed the utility of the machine learning model to generate ingredient mixtures showing effectiveness in shifting a baseline ratio of non-beneficial to beneficial bacterial species to provide cosmetic benefits, here a reduction in axillary malodor.

EXAMPLE 11: In vivo Analysis of Hybrid Cosmetic Stick with 4% (w/w) Blend 83 Effectiveness to Reduce Volatile Organic Compound Generation in Multiple Human Volunteers

[0266] The procedure used above with a single human volunteer in Example 10 was used with 8 additional human volunteers. A mean difference between an amount of volatile organic compounds between left and right axillary regions after use of the hybrid stick formulation with and without addition of Blend 83 at 4% (w/w) Hybrid Cosmetic Stick was evaluated.

[0267] Results: There was a significant difference in the amounts of volatile organic compounds present in each volunteer’s left and right axillary regions after use of the 4% (w/w) Blend 83 Hybrid Cosmetic Stick. FIG. 7, shows the number of people who experienced a decrease of 25% or greater of the referenced volatile organic compounds. [0268] The below Table 16 presents the results of most, if not all, of the volatile organic compounds that were identified, along with a general characterization of human smell perception of the odor from the compound, as well as whether the amount was increased or reduced as compared to the control side of each person.

[0269] Table 16: Volatile Organic Compounds Identified by GC/MS when Blend 83 is Used In Vivo Collected from 7 Human Volunteers and Difference in Volatile Organic Compounds as Compared to Control Side

Acetoin- Pleasant, buttery, found in s Not specified § Reduced dairy products like kefir and § yogurt, described as having a s § yogurt odor and a fatty ? § creamy butter taste [ [

* Odor characterization and perception derived from online searches for each volatile organic compound

[0270] From these results, a conclusion was drawn that not only was an amount of malodor in the left axillary area reduced in the subject by use of the containing 4% (w/w) Blend 83 Hybrid Cosmetic Stick, but also that the formulation caused a shift in a ratio of beneficial to non-beneficial bacterial species in the subject’s left axillary area relative to a baseline — or “native” — ratio of axillary bacterial species in the subject. Moreover, there was a marked difference in the types of volatile organic compounds generated in the human volunteers as compared to the control side, with many of the compounds being reduced being associated with odors that humans can find “unpleasant, “pungent,” or the like.

[0271] This Example further confirmed the utility of the methodology herein to generate ingredient mixtures effective to shift baseline ratios of non-beneficial to beneficial bacterial species to provide cosmetic or cosmetic-like modifications to a skin site location of person in need thereof, here a reduction in axillary malodor. Moreover, taking Examples 10 and 11 together, the effectiveness of the processes herein were observed in 8 out of 9 subjects. This indicates that the methodology generated by the inventors are effective to predict ingredient combinations that are not only effective to address in vitro generation of volatile organic compounds that are associated with axillary malodor, but also to reduce malodor in a variety of human subjects when incorporated in an effective amount in a personal care formulation.

EXAMPLE 12: Human Subject Self- Assessment Testing with Blend 83 in Hybrid Cosmetic Stick

[0272] A two-group consumer perception study was conducted in which individuals were instructed to use the Hybrid Cosmetic Stick set out in Table 8 including Blend 83 at 4% (w/w). Each group contained 30 adult participants.

[0273] Before participating in the study, participants were instructed not to use any deodorant or other product on their underarms for three straight days as a “washout period.” Participants were allowed to use an alcohol wipe to clean the underarm area. Participants also reported that underarm smell was a concern for them. Potential participants were excluded if they were regular antiperspirant users. During that time, participants were instructed not to use any other product on their underarms. On Day 4 of the trial, participants were asked to use the Blend 83 4% (w/w) Hybrid Cosmetic Stick after taking a shower in the morning.

[0274] Participants used a test product on Days 5 and 6 and took surveys on those same days. After completing the survey on Day 6, participants concluded their participation in the study. At the end of Day 6, participants were asked questions about their perception of the test products.

[0275] Results: 97% of the participants indicated that the 4% Blend 83 (w/w) Hybrid Cosmetic Stick did not cause irritation in their underarms. 77% of the participants agreed that an amount of underarm odor was decreased with use of the Blend 83 4% (w/w) Hybrid Cosmetic Stick.

[0276] The human subject self-assessment demonstrates that the machine learning methodology that is used to identify potential ingredient mixtures is not only effective to assist in the identification of compositions that can reduce axillary malodor by shifting a ratio of non-beneficial to beneficial bacterial species in in vitro and in vivo testing, but also that the effects can be observed in real-life when personal care formulations including an effective amount of the ingredient mixture are used.

EXAMPLE 13: TEWL/Moisturization Studies of 4% (w/w) Blend 83 Anhydrous Cosmetic Stick

[0277] A test was performed to determine whether a shift in an amount of bacterial species in a user’s axillary region provided any cosmetically relevant effects in addition to a reduction in malodor. It was hypothesized that the increase in an amount of S. epidermidis might provide moisturization benefits to the axillary region. To this end, the Hybrid Cosmetic Stick including 4% Blend 83 was evaluated in a clinical study for moisturization and skin barrier repair properties on 33 female volunteers between the ages of 18 to 65. Inclusion criteria for the trans epidermal water loss (“TEWL”) testing was that participants not use of any skin treatment products on the underarm for three days before the testing began and nor during the study. Participants were also instructed not to wash their underarms for three hours prior to testing.

[0278] Following assessment of non-treatment skin barrier functionality in each volunteer using a Tewameter (Tewamater TM300, Courage and Khazaka Electronics, GmBH, Cologne, DE), a single application of 50 pL of 4% Blend 83 (w/w) was made to an axillary area of each volunteer and spread evenly across with a gloved finger over a 5cm x 5xm marked area. Additional TEWL assessments were performed at baseline, 24 hours and 48 hours following the Blend 83 application. Subjects were required to be in an environmentally controlled room (22°C +/- 2°C/ 45% +/- 5% relative humidity) for approximately 30 minutes before any assessment of TEWL.

[0279] Results: Blend 83 at 4% (w/w) in water produced statistically significantly higher TEWL values (p<0.05) than the Negative Control at baseline, 24 hours and 48 hours following treatment. The results showed a statistically significant 15% and 13% increase in barrier function over 24 and 48 hours, respectively. The results are shown in FIG. 8. Additionally, the data from the TEWL measurements demonstrate that there were no statistically significant differences (p>0.05) between the test site and the untreated site prior to application of Blend 83 to each volunteer’s axillary region, further confirming the validity of the study.

[0280] This Example indicates that the methodology herein has utility not only in identifying ingredient mixtures that can effectively reduce axillary malodor by shifting the ratio of non- beneficial to beneficial bacterial species, but also that ingredient mixtures can provide additional cosmetic benefits to uses of personal care products incorporating the ingredient blends.

EXAMPLE 14: Blend 2 Effectiveness in Reducing Malodorous Compound Formation from Mock Bacterial Community

[0281] To further elucidate the utility of the methodology disclosed herein to assist in the identification of ingredient mixtures effective to change a ratio of beneficial microbial species to non-beneficial microbial species in a human axillary region, Blend 2 as set out in Table 6 above was evaluated for an effectiveness in affecting a ratio of beneficial bacterial species to non-beneficial bacterial species as appearing in a human axillary region.

[0282] At a normalized starting OD600 of 0.06, a mock axillary bacterial community comprised of C. jeikeium (16.67%), C. tuberculostearicum (16.67%), C. striatum (16.67%), S. hominis (10%) , C. acnes (20%) and S. epidermidis (20%), in the proportions stated in the parentheses, was incubated for 16h at 37 °C in SLM and lOmM 3M2H-Glu (malodor precursor). After incubation, malodorous compound production as an aggregated amount of 3M2H-related compounds generated from the bacterial species in the mock community was measured via GC-MS.

[0283] Results: As shown in FIG. 9, Blend 2 resulted in a reduction in 3M2H-related compounds that was similar to Blend 83, and both of these ingredient mixtures reduced formation of these malodorous compounds in comparison to the Control.

[0284] This result further confirms the utility of the methodology herein to identify ingredient compositions that have potential for the modification of an amount of axillary malodor in humans.

EXAMPLE 15: Comparison of Blend 83 and Blend 2 Effectiveness in Reducing Malodorous Compound Formation from Mock Bacterial Community Comprising Both Corynebacterium sp. and Staphylococcus sp.

[0285] As currently understood, axillary malodor is, in part, caused by S. hominis. An ingredient mixture that exhibited specificity on the group of bacterial species that are the primary cause of axillary malodor independent of genus would be beneficial in a personal care product configured to address this cosmetic need. The machine learning model of the present disclosure was trained to differentiate ingredient effectiveness on individual bacterial species and to predict specificity to each bacterial species. Such predictions were reviewed by a human expert for appropriateness for use in a personal care formulation, as discussed previously.

[0286] Blend 83 and Blend 2 were previously shown to have effectiveness in the reduction of the volatile compounds typically associated with axillary malodor on a bacterial species level, as shown in the above Examples. A further analysis was conducted to determine whether each of these ingredient mixtures demonstrated specificity on a bacterial species level. In this regard, a comparison of the effectiveness of Blend 83 and Blend 2 in reducing axillary malodor-causing compounds was conducted on individual cultures of each of the bacterial species typically associated with the generation of malodorous compounds in the human axillary region, specifically: C. jeikeium, C. striatum, C. tuberculostearicum, and S. hominis, as set out in Example 14. Individual microbes for each of these bacterial species were grown in SLM and 10 mM Benzyl-cysteine (malodor precursor) for 16h at 37 °C and a relative amount of thiol produced therefrom was determined by GC/MS as set out previously.

[0287] Results: As shown in FIGS. 10A-D, Blend 83 and Blend 2 both reduced the relative amounts of thiol production generated from each of the axillary malodor producing microbes. These results directionally indicate that both of these ingredient mixtures generated can exhibit specificity for microbial species known to cause axillary malodor and not just on a bacterial genus level. It can then be hypothesized that the machine learning model can be configured to identify ingredients and ingredient mixtures having selectivity on individual types of bacterial species, such within and among the Corynebacterium sp. and Staphylococcus sp. that do and do not cause axillary malodor.

EXAMPLE 16: qPCR Assay of Specificity of Blend 83 and Blend 2 on Axillary Bacterial Species

[0288] To further evaluate the ability of the methodology herein to identify ingredients and mixtures of ingredients that can exhibit specificity to enhance the growth of beneficial bacterial species in the axillary region and at the same time reduce the growth of non- beneficial bacterial species therein, a Quantitative PCR (“qPCR”) study was conducted. As would be appreciated, qPCR is a method that measures the number of copies of a mRNA region defined by a particular PCR primer(s). Using this method, the 16S amplicon is amplified and the total bacterial count is quantified in each sample to determine total bacterial load.

[0289] For this test, a mock axillary bacterial species community comprised of non-beneficial microbial species as C. jeikeium, C. tuberculostearicum, C. striatum, S. hominis and beneficial bacterial species as C. acnes and S. epidermidis as in Example 14. Moreover, C. acnes is generally considered to be a beneficial bacterial species when present in the human axillary region and, as such, was included in this Example.

[0290] This mock axillary bacterial species community was incubated for 16h in SLM and 10 mM 3M2H-Glu as the malodor precursor. After incubation, malodorous compound production was measured via GC-MS. 500 pL aliquots were taken from each of the culture samples. DNA was extracted following the addition of propidium monazide (which prevents DNA from dead cells to be amplified). qPCR assays for each of the microbes constituting the mock communities were then run to determine the community dynamics of these cultures. [0291] Results: As shown in FIG. 11, Blend 83 and Blend 2 reduce the relative abundance of C. jeikeium, C. tuberculostearicum, and C. striatum. The relative abundance of 5. epidermidis and C. acnes are increased with Blend 83, whereas Blend 2 only affected the relative abundance of C. acnes. The growth of S. hominis was reduced with Blend 83 but not Blend 2 compared to the Control, which directionally indicates that Blend 83 may have some enhanced specificity to increase the growth of S. epidermidis, but not S. hominis. In other words, Blend 83 appears to exhibit prebiotic activity on S. epidermidis, but has only a slight effect, if any, on the growth of S. hominis. Moreover, the increase in growth of C. acnes with Blend 2 as compared to Blend 83 may directionally indicate that the Blend 2 ingredients can enhance the growth of this bacterial species preferentially over the ingredients in Blend 83. In areas where C. acnes may result in problematic skin conditions (e.g., the face), this result may be useful. In other areas where C. acnes may be identified as a beneficial bacteria, the selectivity of Blend 2 on this bacterial species might also be of interest. In short, this Example confirms the ability of the machine learning processes herein to identify ingredient mixtures that can generate selective activity on one or more bacterial species that have been identified as beneficial or non-beneficial in relation to a human body site of interest.

[0292] It is notable that the lesser growth of .S', epidermidis with Blend 83 and/or the increased growth of S. hominis with Blend 2 is not necessarily detrimental to the use thereof in personal care formulations. To the contrary, the ability to identify ingredient mixtures predicted to modify ratios of beneficial microbial species to non-beneficial microbial species in a skin site location can have utility for use in the formulation of personal care formulations. For example, such information can be used to identify ingredients that might have an effect of increasing or decreasing growth of beneficial microbial species and non- beneficial microbial species in a selected human body site of interest. Thus, a formulator can review the results of bacterial species growth as useful information for use in developing new personal care products intended to effect — or not affect — the growth of beneficial microbial species and/or non-beneficial microbial species in a skin site location of interest. For example, observed prebiotic activity (or lack thereof) of an ingredient mixture on either a beneficial microbial species or non-beneficial microbial species as developed from the methodology herein can be evaluated for use in a personal care formulation in regard to an intended cosmetic or cosmetic-like modifications intended to be generated in a skin site location in a human who uses that product as directed.

EXAMPLE 17 : Evaluation of Ingredient Mixtures for Scalp Applications

[0293] Mock Bacterial Species Community and Microbial Species Shifting Model Development: As an initial step, a mock scalp microbial species community was generated to test an effect of ingredient mixtures predicted to have an activity on the specific microbiota that are present the human scalp region. Also, a method to identify a change in one more microbial species after treatment with ingredient mixtures was developed to see whether and to what effect tested ingredient mixtures had on increasing on a mock scalp microbial species community. To this end, information about the microbial species make up of “healthy” scalp and “non-healthy” (i.e., exhibiting redness, irritation, dryness, and/or flaking) scalp microbiota was generated by the inventors from the following literature sources:

• Woo YR, Cho M, Han Y, Lee SH, Cho SH, Lee JD, Kim HS. “Characterization of Distinct Microbiota Associated with Scalp Dermatitis in Patients with Atopic Dermatitis.” J Clin Med., 2022 Mar 21 ; 11(6): 1735.

• Xu, Z., Wang, Z., Yuan, C. et al. “Dandruff is associated with the conjoined interactions between host and microorganisms.” Sci Rep 6, 24877 (2016).

• Chong, Charlotte Elizabeth. “Metagenomic analyses of the scalp microbiome and insights from Staphylococcus capitis genomics.” The University of Liverpool (United Kingdom), 2021.

• Clavaud C, Jourdain R, Bar-Hen A, Tichit M, Bouchier C, Pouradier F, El Rawadi C, Guillot J, Menard-Szczebara F, Breton L, Latge JP, Mouyna I. “Dandruff is associated with disequilibrium in the proportion of the major bacterial and fungal populations colonizing the scalp.” PLoS One. 2013 ;8(3).

• Grimshaw, Sally G., et al. "The diversity and abundance of fungi and bacteria on the healthy and dandruff affected human scalp." PLoS One. 2019; 14 (12).

[0294] For the purposes of assessing the effectiveness of the various system-generated ingredient mixtures on a Mock “Dandruff’ Microbial Species Community was prepared. In this regard, the inventors surveyed the literature in which the microbial species in human scalp had previously been sequenced, and those microbial species that consistently showed up across that sequencing data where averaged in the reported relative abundances of them to generate the Mock “Dandruff’ Microbial Species Community. The make-up of the scalp community generated for testing of the ingredient mixtures for scalp was as follows:

[0295] Table 17: Scalp Skin Site Beneficial and Non-Beneficial Microbial Species

[0296] Model Scalp Like Medium: A model scalp growth medium for use in testing an efficacy of the system-generated ingredient mixtures was developed with the following ingredients.

[0297] Table 18: Ingredients for Scalp-like Medium

[0298] Table 19: Procedure for preparing scalp-like media, as a combination of modified RPMI media and artificial sebum

[0299] A number of ingredient mixtures predicted to have effectiveness in modifying a ratio of beneficial microbial species to non-beneficial microbial species in the Mock “Dandruff’ Microbial Species Community were reviewed for suitability for use in a personal care formulation appropriate for use as a rinse off shampoo, since this is a primary vehicle for treatment of dandruff-like conditions using over-the-counter formulations. Ingredient mixtures denoted Blend 15, Blend 16, and Blend 162 from Table 20 below were selected for testing by human review of the ingredient mixtures predicted to have relevant scalp prebiotic activity by the machine learning system. Selection was based on a number of factors including, for example, availability of the mixture ingredients, likely formulation problems, etc.

[0300] It should nonetheless be noted that an ingredient mixture that cannot suitably be delivered from a rinse off shampoo formulation could also have utility in determining whether an ingredient mixture may have utility in modifying an amount of a beneficial microbial species or a non-beneficial microbial species that may be present in a skin site location. For example, a serum or other leave in formulation might be appropriate in some use cases.

[0301] Table 20: Ingredient Mixtures Predicted to have Activity on Scalp Microbial Species

[0302] A simulated rinse-off shampoo procedure was used to evaluate the Blends from Table 20 above. To summarize, each Mock “Dandruff’ Microbial Species Community was washed with 1% of each blend ingredient mixture in Scalp-Like Medium. For each wash, the test ingredient/Scalp-Like Medium mixture was mixed and then the samples were centrifuged. Fresh/pre-warmed Scalp-Like Media was then added to each tube for the following 12h culture. The incubation was at 35 °C.

[0303] Each of ingredient mixtures from Table 20 were tested in Scalp-Like Medium with the Mock “Dandruff’ Microbial Species Community as set out above using redox dye to monitor metabolic activity allowing one to evaluate the impact of the blend to the metabolic activity of the tested microbial species. This procedure is illustrated in FIG. 12. As discussed herein, screening of these selected ingredient mixtures were assessed to determine whether the growth of beneficial microbial species is enhanced with an ingredient mixture such that a ratio of beneficial microbial species to non-beneficial microbial species in the direction of reducing a relative amount of non-beneficial microbial species as compared to the initial Mock “Dandruff’ Microbial Species Community control.

[0304] The effect of Blend 162, Blend 15 and Blend 16 on an amount of bacterial species in the Mock “Dandruff’ Microbial Species Community was evaluated for an effect on metabolic activity. 500 pL 1% of each blend was incorporated in 4.5 mL Mock “Dandruff’ Microbial Species Community in the scalp-like medium (about 10⁶ colony forming unit per ImL).

[0305] Results: As shown in FIG. 13, the positive control of zinc pyrithione — as an active ingredient for use in dandruff shampoo formulations — reduces dandruff associated microbial species, but also greatly reduce beneficial microbial species. Blends 162, 15, and 16 each demonstrate a reduction in not only dandruff-associated microbial species, but they also maintain or promote beneficial microbial species that are present in human scalp areas. [0306] This Example further demonstrates that the ingredient mixtures and associated testing can effectively operate to identify compositions that can modify a ratio of beneficial microbial species to non-beneficial microbial species, here with the example being a human scalp area.

EXAMPLE 18: Evaluation of Blends 15, 16, and 162 in Shampoo Base

[0307] As would be understood, the utility of an ingredient mixture as an active ingredient can be significantly affected by the ability to deliver enough of the active ingredient from a personal care formulation. The effectiveness of Blends 15, 16, and 162 were thus evaluated in a commercial shampoo base branded as “Thick Shampoo and Body Wash” (Essential Labs, Portland, OR). As reported on the manufacturer’s website (https://essentiallabs.com/collections/shampoo-body-wash/products/thick-shampoo-body- wash, retrieved 11/21/2023), the product can be customized to fit specific needs. It is understood that this shampoo base can be used for the addition of custom-selected ingredients. This shampoo was sold without fragrance, meaning that any non-microbial activity that might be associated therewith would not be present in this formulation. The amounts of each ingredient were not available due to the commercial nature of this product. The ingredients of this shampoo — denoted as “Shampoo A” in this Example were reported as follows, with the ingredient functions incorporated for reference. 1% (w/w) of each ingredient mixture was added to Shampoo A.

[0308] Table 21: Shampoo A Ingredients and Function

[0309] A human use study was conducted to assess an effectiveness of each of the ingredient mixtures when delivered from the Shampoo A formulation in typical consumer use cases for a two-week period.

[0310] Adult (18-64) male and female participants who self-reported at least one scalp issue (e.g., redness, irritation, dryness, and/or flakiness) were recruited for this study. Each participant was instructed to use a respective shampoo formulation 3 times a week for 2 weeks. At the end of each week, each participant was asked to rate their scalp condition by providing “Yes” or “No” answers to the following:

• Did you observe a decrease in flakes and/or dandruff?

• Does your scalp feel less irritated, itchy, dry, tight, tender, and/or uncomfortable?

• Does your scalp appear to be less red and/or inflamed?

[031 1] Results: After 2 weeks of use, each of Blends 15, 16, and 162 performed better than the Placebo/Shampoo A only. The best overall performance was observed for Blend 162. The results can be summarized as follows for each ingredient mixture as delivered at 1 % (w/w) from a Shampoo A base, where the results were compared to the Placebo/Shampoo A base only.

[0312] Table 22: Consumer Preference Results for Blends 15, 16, and 162 Delivered from Shampoo A Base at 1% (w/w) as Compared to Placebo/Shampoo A Only

[0313] A summary of the collected results of this consumer preference study are shown in FIG. 14. From the results of this consumer preference study, Blend 162 was selected for additional in vivo testing. EXAMPLE 19: Metabolic Activity Study of Effectiveness Of Blend 162 With Common OTC Dandruff Actives On Mock “Dandruff’ Microbial Species Community

[0314] The ingredient mixture denoted Blend 162 was examined for the effect on a Mock “Dandruff’ Microbial Species Community at 1.0% with and without 1.8% salicylic acid (“SA”), as a common over-the-counter (“OTC”) scalp care active ingredient. A comparison was also made in relation to SA alone and piroctone olamine, another common over-counter- ingredient for the treatment of dandruff. The procedure used for this Example was as set out in Example 17 above.

[0315] Results: As shown in FIG. 15 Blend 162 was effective in modifying the amount of non-beneficial microbial species in the Mock “Dandruff’ Microbial Species Community both with and without 1.8% SA. As compared to the piroctone olamine control, which negatively impacted both the non-beneficial microbial species and the beneficial microbial species, Blend 162 at 1.0% with no SA appeared to exhibit specificity on the non-beneficial microbial species in the community, while appearing not to substantially affect the presence of the beneficial microbial species.

[0316] This Example further indicates that the ingredient mixture denoted Blend 162 is effective at 1.0% in inhibiting dandruff-associated Malassezia and S. capitis while also promoting the growth of S. epidermidis, C. acnes, and C. granulosiim, three beneficial- representative scalp microbes in a Mock “Dandruff’ Microbial Species Community as compared to SA and piroctone olamine controls. This again shows the utility of the methodology herein to identify ingredient mixtures that can effectively shift the microbiota present in a skin site location — here, the scalp area-by selectively acting on non-beneficial microbial species, while at the same time allowing the beneficial microbial species to remain viable at the location.

EXAMPLE 20: In Vivo Testing of Blend 162 in Shampoo A Base in 7 Human Volunteers

[0317] A 20-day study of an effectiveness of Blend 162 as delivered at 1% from Shampoo A in 7 adult male and female study participants (male & female) was conducted. After a 5 -day washout period in which no product was used on their hair, each participant was instructed to use the formulation every other day. Swabs of their scalp were taken at different times for indicators of dandruff using the peroxidation analysis and sequencing set out above. Imaging of each person’s scalp areas was conducted using a Dermatoscope, which is a handheld instrument that can magnify skin up to 10 times. They are used to examine skin and can record images for future comparison. Participants also self-reported their perceptions of improvement in their scalp condition.

[0318] Results: As shown by FIG. 16A, 5/7 participants demonstrated a microbiome shift both compared to baseline and compare to after washout. The shift was in general seen by a reduction in the abundance of A capitis and an increase in the abundance of 5. epidermidis. Additionally, as shown in FIG. 16B, a reduction in the abundance of Malassezia species was observed for all participants compared to both baseline and after washout. Presumably due to the shift of the scalp microbiome compositions of the participants, 5/7 participants have decreased SQ:SQOOH after washout, as shown in FIG. 17). This result indicated that these participants were at least partially symptomatic of scalp-related health issues after they stopped use of haircare products for 5 days. Notably, 6/7 participants exhibited increased SQ:SQOOH at the end of the study (8 washes). This indicates that the Shampoo A with 1% of Blend 162 improved the scalp condition.

[0319] Scalp images from 3 of the human volunteers using the Dermatoscope are shown in FIG. 18. As apparent in the original color photographs, each volunteer exhibited a lower level of redness after 8 washes as compared to at least the Day 0 results, which was after the volunteers stopped using their normal haircare treatments.

[0320] The participants also reported results that are consistent with the improvements of in scalp condition after using 1% Blend 162 in Shampoo A as shown in Table 23 below.

[0321] Table 23: Participants Self-Reported Perceptions of Blend 162 as Delivered from Shampoo A Base

• A= No response

• B= No effect

• C= Neutral effect

• D= Positive effect

[0322] Collectively, the results of this Example shows the effectiveness of the Blend 162 as delivered from Shampoo Base A at 1% (w/w).

[0323] The Examples hereinabove demonstrate the efficacy of a high throughput screening method to generate preliminary predictions of activity of ingredients to shift a starting ratio of beneficial microbial species to non-beneficial microbial species in a human skin site location to a different. Testing of ingredient mixtures selected on the basis of individual activity on relevant beneficial microbial species and beneficial microbial species as needed for a specific skin care product applications as set out hereinbelow illustrates how various ingredient mixtures configured from data derived from a large set of data for ingredient activity on individual microbial species relevant to the cosmetic or cosmetic-like conditions of a human’s skin can be used to generate personal care formulations effective to impart benefits thereto. In this regard, when the ingredient mixtures incorporate cosmetic-appropriate ingredient selections and where prebiotic activity of the mixtures that can be appropriately formulated into personal care products having an acceptability to a consumer, use thereof by consumers can impart a cosmetic or cosmetic -like modification to a skin site location in need thereof.

[0324] Also, information associated with the selection and/or deselection of a systemgenerated ingredient mixture and the results from in vitro screening and, if relevant, in vivo screening of an ingredient mixture can be incorporated in the machine learning model to improve subsequent prebiotic activity predictions therefrom.

Claims

What is claimed is:

1. A method of identifying an ingredient mixture effective to modify a cosmetic or cosmetic-like condition in a skin site location, the method comprising: a. contacting a test sample containing the ingredient mixture with a formulation of mock microbial species community that is configured to simulate a baseline ratio of at least one beneficial microbial species and at least one non-beneficial microbial species in the skin site location in a real-life person, and b. selecting the test sample, optionally for generating a personal care formulation, when the test sample exhibits: i. prebiotic activity on the at least one beneficial microbial species; and ii. non-prebiotic activity on the at least one non-beneficial microbial species.

2. The method of claim 1, further comprising selecting the skin site location prior to step (a), wherein the condition is associated with an excess of an amount of the at least one non-beneficial microbial species to an amount of the at least one beneficial microbial species present in the skin site location in a real-life person.

3. A method of identifying an ingredient mixture effective to modify a cosmetic or cosmetic-like condition in a skin site location, the method comprising: a. selecting a skin site location of interest for generation of a personal care formulation, wherein: i. the formulation is configured for the skin site location; ii. the condition is associated with an excess of an amount of at least one non-beneficial microbial species to an amount of at least one beneficial microbial species present in the skin site location in a real-life person; b. generating a mock microbial species community configured to determine an activity of each of a plurality of ingredient mixtures to affect an amount of activity of each of at the least one beneficial microbial species and the at least one non-beneficial microbial species from a first time to a second time, wherein the mock bacterial community is configured to simulate a baseline ratio of the at least one beneficial microbial species and the at least one non- beneficial microbial species in the skin site location in the real-life person; c. preparing a plurality of test samples each comprising an amount of: i. the ingredient mixtures; ii. the mock microbial species community; and iii. a composition configured to simulate a biological material present at the skin site location; d. evaluating each the test samples for a presence or absence of an increase in a ratio of the at least one beneficial microbial species to the at least one non- beneficial microbial species, wherein the ratio is determined by comparing an amount of each of the beneficial microbial species and non-beneficial microbial species present in each test sample at each of a start and an end of an incubation step; and e. generating a determination whether one or more of the of the plurality of ingredient mixtures exhibits each of: i. prebiotic activity on the at least one beneficial microbial species; and ii. non-prebiotic activity on the at least one non-beneficial microbial species. The method of claim 3, further comprising generating information associated with whether one or more of the plurality of ingredient mixtures has a presence or absence of effectiveness in modifying the cosmetic or cosmetic-like condition in the real-life person when the person uses a personal care formulation incorporating an effective amount of one of the plurality of ingredient mixtures from a first time to a second time. The method of claim 3, further comprising: a. selecting an ingredient mixture determined to exhibit prebiotic activity on the at least one beneficial microbial species; and b. incorporating an effective amount of the selected ingredient mixture in a personal care formulation, wherein the formulation is configured with a plurality of cosmetically appropriate ingredients selected to allow delivery of a majority of the ingredient mixture’s prebiotic activity to the at least one beneficial microbial species when the formulation is applied to a body site location in need of modification in the condition in a real-life person. The method of claim 3, wherein an amount of metabolic activity of each of the at least one beneficial microbial species and the at least one non-beneficial microbial species are determined at the end of the incubation step, thereby providing information associated with an effectiveness of each ingredient mixture on modifying an amount of growth of each of the beneficial microbial species and non-beneficial microbial species during the incubation step. The method of claim 3, wherein an amount and a type of a plurality of volatile organic compounds present in one or more of the test samples is determined at an end of the incubation step, and the method further comprises comparing the type and amount of the plurality of volatile organic compounds generated at the end of the incubation step with a control test sample that includes an amount of the mock bacterial community and the composition without addition of any of the plurality of ingredient mixtures. The method of claim 3, wherein a plurality of ingredient mixtures are determined to exhibit prebiotic activity on the at least one beneficial microbial species, and the method further comprises: a. selecting an ingredient mixture for testing of an effectiveness in modifying the cosmetic or cosmetic-like condition when the mixture is incorporated in a personal care formulation configured for use in the skin site location; b. preparing the personal care formulation incorporating an amount of the selected ingredient mixture; c. selecting a plurality of human volunteers each having a need for modification of the cosmetic or cosmetic-like condition on the body site location; and d. evaluating an effectiveness of the personal care formulation in modifying the condition when each person applies an amount of the personal care formulation at least once daily from a first date to an assessment date. The method of claim 7, further comprising: a. obtaining from each of the plurality of human volunteers a self-assessment of a presence or absence of a change in the cosmetic condition at the skin site location from the first date to the assessment date; and b. generating information associated with a preference of each of the plurality of human volunteers for the personal care formulation. method of any one of claims 1 to 9, wherein: a. the body site location is an axillary region of a first person; b. an ingredient mixture exhibiting each of prebiotic activity on the at least one beneficial microbial species and non-prebiotic activity on the at least one non- beneficial microbial species is selected for incorporation in a personal care formulation configured for application to the first-person axillary region; c. the first-person axillary region is associated with a baseline level of beneficial microbial species and non-beneficial microbial species in the when the person at an end of an antiperspirant and deodorant product washout period; and d. the cosmetic or cosmetic-like modification comprises a decrease in an amount of axillary malodor in the first person axillary region when the person applies the personal care formulation comprising an effective amount of the selected ingredient mixture to their axillary region at least once a day from a first date proximate to the washout period to an axillary malodor assessment date. method of claim 10, wherein: a. the at least one beneficial microbial species comprises Staphylococcus epidermidis and the at least one non-beneficial microbial species comprises Corynebacterium jeikeium ; and b. the decrease in the amount of axillary malodor in the first person is associated with an increase in a ratio of the Staphylococcus epidermidis to Corynebacterium jeikeium as determined from the first date to the axillary malodor assessment date. method of claim 10, wherein: a. the formulation comprises a plurality of cosmetically appropriate ingredients; and b. the ingredients are selected to allow a majority of the prebiotic activity of the selected ingredient mixture to be delivered to the body site location when the real-life human applies the formulation to their axillary region. The method of claim 12, wherein: a. the first-person axillary region comprises a native amount of the at least one beneficial microbial species and the at least one non-beneficial microbial species; and b. the ingredients in the formulation other than the selected ingredient mixture substantially do not exhibit any prebiotic or non-prebiotic activity on the at least one beneficial microbial species and at least one non-beneficial microbial species that are natively present in the first-person axillary region. The method of claim 10, wherein the personal care formulation is configured in the form of a cosmetic stick. The method of claim 10, wherein the selected ingredient mixture is included in the personal care formulation at from about 0.5 to about 5% (w/w) of the formulation. The method of claim 10, wherein the cosmetic or cosmetic-like modification further comprises a decrease in an amount of trans epidermal water loss in the axillary region from the first date to the assessment date. The method of claim 10, wherein the desired cosmetic or cosmetic-like modification comprises a reduction in a generation of an amount of thiol-containing volatile organic compounds as measured from the first date to the assessment date. The method of any one of claims 1 to 3, wherein: a. the body site location is a scalp region of a first person; b. an ingredient mixture exhibiting each of prebiotic activity on the at least one beneficial microbial species and non-prebiotic activity on the at least one non- beneficial microbial species is selected for incorporation in a personal care formulation configured for application to the first-person scalp region; c. the first-person scalp region is associated with a baseline level of beneficial microbial species and non-beneficial microbial species when the person at an end of a haircare product washout period; and d. the cosmetic or cosmetic-like modification comprises a decrease in an amount of one or more of scalp redness, scalp dryness, scalp irritation, or scalp flaking in the first person scalp region when the person applies the personal care formulation comprising an effective amount of the selected ingredient mixture to their scalp region at least once a day from a first date proximate to the washout period to a scalp condition assessment date. method of claim 18, wherein: a. the at least one beneficial microbial species comprises Staphylococcus epidermidis and the at least one non-beneficial microbial species comprises Staphylococcus capitis and Malassezia restricts, and b. the decrease in the amount of one or more of scalp redness, scalp dryness, scalp irritation, or scalp flaking in the first-person scalp region is associated with an increase in a ratio of the Staphylococcus epidermidis to each of the Staphylococcus capitis and M. restricta as determined from the first date to the scalp condition assessment date. method of claim 18, wherein: a. the formulation comprises a plurality of cosmetically appropriate ingredients; and b. the ingredients are selected to allow a majority of the prebiotic activity of the selected ingredient mixture to be delivered to the body site location when the real-life human applies the formulation to their scalp region. method of claim 18, wherein: a. the first person scalp region comprises a baseline amount of the at least one beneficial microbial species and the at least one non-beneficial microbial species; and b. the ingredients in the formulation other than the selected ingredient mixture substantially do not exhibit any prebiotic or non-prebiotic activity on the at least one beneficial microbial species and at least one non-beneficial microbial species that are present in the baseline amount in the first-person scalp region. The method of claim 18, wherein: a. the scalp comprises an amount of squalene at the end of the washout period, wherein the squalene is subject to peroxidation; and b. the cosmetic or cosmetic-like modification is associated with a reduction in an amount of peroxidation of squalene as present in the scalp as measured from the first date proximate to the washout period to the scalp condition assessment date. The method of claim 18, wherein the personal care formulation is configured as a rinse-off shampoo formulation or a leave in serum. The method of claim 18, wherein the selected ingredient mixture is included in the personal care formulation at from about 0.5 to about 5% (w/w) of the formulation. A personal care formulation comprising: a. Blend 83 or Blend 2; or b. Blend 15, Blend 16, or Blend 162; and a cosmetically acceptable excipient; wherein when present (i) Blend 83 or Blend 2 is present in an amount between 1% (w/w) and 10% (w/w), inclusive, between 1% (w/w) and 7.5% (w/w), inclusive, or between 1% (w/w) and 10% (w/w), inclusive, (ii) Blend 15, Blend 16, or Blend 162 is present in an amount between 0.01% (w/w) and 5% (w/w), inclusive, between 0.01% (w/w) and 2.5% (w/w), inclusive, or between 0.01% (w/w) and 1.5% (w/w), inclusive. The personal care formulation of claim 25, produced by a method of any one of claims 1 to 17 or 19 to 24.