Bacterial strains for skin health

Abstract

A hydrogel comprising a lysate or extract from Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1) or Enterococcus Avium strain HL-95 (SEQ ID NO: 2) and methods of using the same for skin health, skin protection, and wound healing.

Description

Attorney Docket No.11001-206WO1 BACTERIAL STRAINS FOR SKIN HEALTH CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 608,636, filed December 11, 2023, which is incorporated by reference herein in its entirety. GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. W81XWH-18- PRARP AZ180098 awarded by the Department of Defense and Grant Nos. R56AG069676, R56AG064075, R01AG071762, R21AG072379, R21DE032197, U01AG076928 and R21AG082164 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND

[0003] Wound healing is a complex and dynamic process that involves a series of overlapping phases, including hemostasis, inflammation, proliferation, and tissue remodeling [1]. Effective wound healing requires the coordinated actions of various cells, cytokines, and growth factors to restore the integrity of the skin barrier and tissue architecture [2]. Any disruption to these processes can lead to delayed healing, chronic wounds, or excessive scar formation, which presents a significant clinical challenge [3]. In recent years, the exploration of therapies such as bacterial extracts and postbiotics has gained momentum as potential agents to accelerate and enhance wound healing, addressing some of these challenges [4].

[0004] Thus, there exists a need for additional wound healing therapies, including bacterial lysates and postbiotics. These needs and others are at least partially satisfied by the present disclosure. SUMMARY

[0005] In an aspect, provided is a hydrogel including a lysate or extract from Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1).

[0006] In another aspect, provided is a hydrogel including a lysate or extract from Enterococcus Avium strain HL-95 (SEQ ID NO: 2).Attorney Docket No.11001-206WO1

[0007] In yet another aspect, provided is a method of enhancing or promoting wound healing, the method including topically administering a lysate or extract from Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1) to wounded, damaged, or inflamed skin.

[0008] In yet still another aspect, provided is a method of enhancing or promoting wound healing, the method including topically administering a lysate or extract from Enterococcus Avium strain HL-95 (SEQ ID NO: 2) to wounded, damaged, or inflamed skin.

[0009] In yet still another aspect, provided is a method of skin protection, the method including topically administering a lysate or extract from Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1) to skin before exposure to a wounding agent (e.g., UV radiation).

[0010] In yet still another aspect, provided is a method of skin protection, the method including topically administering a lysate or extract from Enterococcus Avium strain HL-95 (SEQ ID NO: 2) to skin before exposure to a wounding agent (e.g., UV radiation).

[0011] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims. BRIEF DESCRIPTION OF DRAWINGS

[0012] FIGURES 1A-1C depict screening of HL-95 and HL-198 as most effective strains. FIG.1A shows the flow of work represented the screening of most effective strains among 240 different strains for wound healing. FIG. 1B represents the top 30 selected strains screened from 240, and FIG.1C represents the top 7 strain based on scratch assay on BJ cell line.

[0013] FIGURES 2A-2I depict the effect of HL-95 and HL-198 on time-dependent re- epithelialization and on reducing inflammatory markers and ROS production in in vitro wound model. FIGS. 2A-2B represent HL-95 and HL-198 treatment increased time dependent cells migration during scratch assay. FIG. 2C depicts production of ROS which was analysed by H2DCFDA assay and is reduced by HL-95 and HL-198. FIG. 2D represents the MMP-9 immunofluorescence increased in control which was attenuated following HL-95 and HL-198 treatment. FIGS.2E-2H show expression of MMP-9 and inflammatory cytokines such as Tnf- ^, Il-6 and Il-1^ respectively, which was decreased following HL-198 treatment. FIG. 2I represent increased fibrillin expression following treatment with HL-95 and HL-198. Fibrillin is a promoter of collagen 3 that is good for reducing scar formation while wound healing is taking place. ^Attorney Docket No.11001-206WO1

[0014] FIGURES 3A-3G show that HL-198 potentiates wound healing and reducing expression of MMP-9 and inflammatory markers during in vivo wound model. FIGS. 3A-3B represent HL-95 HL-198 and betadine treatment increased time dependent reduction in wound area. FIG. 3C represents the MMP-9 immunofluorescence increased in control which was attenuated following HL-95 HL-198 and betadine treatment. FIGS. 3D-3G show expression of inflammatory cytokines such as Tnf-^, Il-6 and Il-1^ respectively, which was decreased following HL-198 and betadine treatment.

[0015] FIGURES 4A-4D show that HL-95 and HL-198 promote epithelization and collagen formation observed by H&E staining and Hydroxyproline assay. FIGS. 4A-4C represent HL-95, HL-198 and betadine treatment increased epidermis formation. FIG. 4D represents the Masson’s trichrome staining as a marker of the collagen fibers, which appear blue and increased following treatment with HL-95, HL-198 and betadine.

[0016] FIGURES 5A-5B show in vitro skin protection against UV exposed model. FIGURES 5C-5D show therapeutic effect against UV exposed model. In FIGS.5A-5B, cells were first treated with different doses of HL-198 and then exposed to UV radiation for 30 min to evaluate the protective effect of HL-198. Treatment with different doses of HL-198 for 24 hours demonstrated a dose-dependent protective effect. The lowest dose (50 µg / mL) did not show protection, while higher doses of HL-198 (100 and 200 µg / mL) significantly reduced UV-induced damage (p < 0.01). However, in FIGS.5C-5D, the cells were first exposed to UV radiation for 30 min and then treated with HL-198 for 24 h to evaluate the therapeutic effect of HL-198. The results represent the highest dose (200 µg / mL) reduced UV-exposed damage to cell and this effect is comparable to non-UV-exposed group. These findings suggest that HL- 198 effectively mitigates UV-induced cellular damage, with its efficacy increasing with dosage. DETAILED DESCRIPTION

[0017] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. 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. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. ^Attorney Docket No.11001-206WO1 DEFINITIONS

[0018] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0019] 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.

[0020] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0021] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0022] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, 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 ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about ^Attorney Docket No.11001-206WO1 x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0023] It is to be understood that 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 numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub- ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0024] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” 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 ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. 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.

[0025] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and ^Attorney Docket No.11001-206WO1 type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

[0026] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0027] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0028] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment ^Attorney Docket No.11001-206WO1 may be varied for example by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0029] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0030] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0031] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0032] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0033] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving ^Attorney Docket No.11001-206WO1 the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0034] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0035] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect. HYDROGELS

[0036] In an aspect, provided is a hydrogel including a lysate or extract from Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1). The whole genome of Lactobacillus Argentoratensis strain HL-198 can be found under GenBank Accession No. SRX27032767.

[0037] In some aspects, the hydrogel can include hydroxypropyl cellulose. In some aspects, the hydrogel can be crosslinked with citric acid.

[0038] In some aspects, the hydrogel can be prepared from a solution including hydrogel precursor and the lysate or extract (for example, the solution can be crosslinked or cured to form the hydrogel). In some aspects, the solution can include about 5 µg / mL or more of the lysate or extract (e.g., about 10 µg / mL or more, about 15 µg / mL or more, about 20 µg / mL or more, about 25 µg / mL or more, about 30 µg / mL or more, about 35 µg / mL or more, about 40 µg / mL or more, about 45 µg / mL or more, about 50 µg / mL or more, about 60 µg / mL or more, about 70 µg / mL or more, about 80 µg / mL or more, about 90 µg / mL or more, about 100 µg / mL or more, about 110 µg / mL or more, about 120 µg / mL or more, about 130 µg / mL or more, about 140 µg / mL or more, about 150 µg / mL or more, about 160 µg / mL or more, about 170 µg / mL or more, about 180 µg / mL or more, about 190 µg / mL or more, about 200 µg / mL or more, about 225 µg / mL or more, about 250 µg / mL or more, about 275 µg / mL or more, about 300 µg / mL or more, about 325 µg / mL or more, about 350 µg / mL or more, about 375 µg / mL or more, about 400 µg / mL or more). In some aspects, the solution can include about 400 µg / mL or less of the lysate or extract (e.g., about 375 µg / mL or less, about 350 µg / mL or less, about 325 µg / mL or less, about 300 µg / mL or less, about 275 µg / mL or less, about 250 µg / mL or less, about 225 µg / mL or less, about 200 µg / mL or less, about 190 µg / mL or less, about 180 µg / mL or less, ^Attorney Docket No.11001-206WO1 about 170 µg / mL or less, about 160 µg / mL or less, about 150 µg / mL or less, about 140 µg / mL or less, about 130 µg / mL or less, about 120 µg / mL or less, about 110 µg / mL or less, about 100 µg / mL or less, about 90 µg / mL or less, about 80 µg / mL or less, about 70 µg / mL or less, about 60 µg / mL or less, about 50 µg / mL or less, about 45 µg / mL or less, about 40 µg / mL or less, about 35 µg / mL or less, about 30 µg / mL or less, about 25 µg / mL or less, about 20 µg / mL or less, about 15 µg / mL or less, about 10 µg / mL or less, about 5 µg / mL or less).

[0039] It is considered that the solution can include an amount of the lysate or extract ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the solution can include from about 5 µg / mL to about 400 µg / mL of the lysate or extract (e.g., from about 10 µg / mL to about 375 µg / mL, from about 15 µg / mL to about 350 µg / mL, from about 20 µg / mL to about 325 µg / mL, from about 25 µg / mL to about 300 µg / mL, from about 30 µg / mL to about 275 µg / mL, from about 35 µg / mL to about 250 µg / mL, from about 40 µg / mL to about 225 µg / mL, from about 45 µg / mL to about 200 µg / mL, from about 50 µg / mL to about 190 µg / mL, from about 60 µg / mL to about 180 µg / mL, from about 70 µg / mL to about 170 µg / mL, from about 80 µg / mL to 160 µg / mL, from about 90 µg / mL to about 150 µg / mL, from about 100 µg / mL to about 140 µg / mL, from about 110 µg / mL to about 130 µg / mL, from about 5 µg / mL to about 120 µg / mL, from about 10 µg / mL to about 110 µg / mL, from about 15 µg / mL to about 100 µg / mL, from about 20 µg / mL to about 90 µg / mL, from about 25 µg / mL to about 80 µg / mL, from about 30 µg / mL to about 70 µg / mL, from about 35 µg / mL to about 60 µg / mL, from about 40 µg / mL to about 50 µg / mL, from about 120 µg / mL to about 400 µg / mL, from about 120 µg / mL to about 375 µg / mL, from about 130 µg / mL to about 350 µg / mL, from about 140 µg / mL to about 325 µg / mL, from about 150 µg / mL to about 300 µg / mL, from about 160 µg / mL to about 275 µg / mL, from about 170 µg / mL to about 250 µg / mL, from about 180 µg / mL to about 225 µg / mL, from about 190 µg / mL to about 200 µg / mL).

[0040] In some aspects, the lysate or extract can include homogenized cellular components of Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1).

[0041] In some aspects, the hydrogel can further include betadine containing 10% povidone-iodine. The hydrogel can include betadine in any suitable amount. For example, in some aspects, the hydrogel can include about 0.01 wt% betadine or more (e.g., about 0.02 wt% or more, about 0.03 wt% or more, about 0.04 wt% or more, about 0.05 wt% or more, about 0.1 wt% or more, about 0.15 wt% or more, about 0.2 wt% or more, about 0.25 wt% or more, about 0.5 wt% or more, about 0.75 wt% or more, about 1 wt% or more, about 1.5 wt% or more, about 2 wt% or more, about 2.5 wt% or more, about 3 wt% or more, about 3.5 wt% or more, about 4 ^Attorney Docket No.11001-206WO1 wt% or more, about 4.5 wt% or more, about 5 wt% or more, about 6 wt% or more, about 7 wt% or more, about 8 wt% or more, about 9 wt% or more, about 10 wt% or more). In some aspects, the hydrogel can include about 10 wt% betadine or less (e.g., about 9 wt% or less, about 8 wt% or less, about 7 wt% or less, about 6 wt% or less, about 5 wt% or less, about 4.5 wt% or less, about 4 wt% or less, about 3.5 wt% or less, about 3 wt% or less, about 2.5 wt% or less, about 2 wt% or less, about 1.5 wt% or less, about 1 wt% or less, about 0.75 wt% or less, about 0.5 wt% or less, about 0.25 wt% or less, about 0.2 wt% or less, about 0.15 wt% or less, about 0.1 wt% or less, about 0.05 wt% or less, about 0.04 wt% or less, about 0.03 wt% or less, about 0.02 wt% or less, about 0.01 wt% or less).

[0042] It is considered that the hydrogel can include an amount of betadine ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the hydrogel can include from about 0.01 wt% to about 10 wt% betadine (e.g., from about 0.02 wt% to about 9 wt%, from about 0.03 wt% to about 8 wt%, from about 0.04 wt% to about 7 wt%, from about 0.05 wt% to about 6 wt%, from about 0.1 wt% to about 5 wt%, from about 0.15 wt% to about 4.5 wt%, from about 0.2 wt% to about 4 wt%, from about 0.25 wt% to about 3.5 wt%, from about 0.5 wt% to about 3 wt%, from about 1 wt% to about 2.5 wt%, from about 1.5 wt% to about 2 wt%, from about 0.01 wt% to about 2 wt%, from about 0.02 wt% to about 1.5 wt%, from about 0.03 wt% to about 1 wt%, from about 0.04 wt% to about 0.75 wt%, from about 0.05 wt% to about 0.5 wt%, from about 0.1 wt% to about 0.25 wt%, from about 0.15 wt% to about 0.2 wt%, from about 1.5 wt% to about 10 wt%, from about 2 wt% to about 9 wt%, from about 2.5 wt% to about 8 wt%, from about 3 wt% to about 7 wt%, from about 3.5 wt% to about 6 wt%, from about 4 wt% to about 5 wt%).

[0043] In another aspect, provided is a hydrogel including a lysate or extract from Enterococcus Avium strain HL-95 (SEQ ID NO: 2). The whole genome of Enterococcus Avium strain HL-95 can be found under GenBank Accession No. SRX27032766.

[0044] In some aspects, the hydrogel can include hydroxypropyl cellulose. In some aspects, the hydrogel can be crosslinked with citric acid.

[0045] In some aspects, the hydrogel can be prepared from a solution including hydrogel precursor and the lysate or extract (for example, the solution can be crosslinked or cured to form the hydrogel). In some aspects, the solution can include about 5 µg / mL or more of the lysate or extract (e.g., about 10 µg / mL or more, about 15 µg / mL or more, about 20 µg / mL or more, about 25 µg / mL or more, about 30 µg / mL or more, about 35 µg / mL or more, about 40 µg / mL or more, about 45 µg / mL or more, about 50 µg / mL or more, about 60 µg / mL or more, about 70 µg / mL or more, about 80 µg / mL or more, about 90 µg / mL or more, about 100 µg / mL ^Attorney Docket No.11001-206WO1 or more, about 110 µg / mL or more, about 120 µg / mL or more, about 130 µg / mL or more, about 140 µg / mL or more, about 150 µg / mL or more, about 160 µg / mL or more, about 170 µg / mL or more, about 180 µg / mL or more, about 190 µg / mL or more, about 200 µg / mL or more, about 225 µg / mL or more, about 250 µg / mL or more, about 275 µg / mL or more, about 300 µg / mL or more, about 325 µg / mL or more, about 350 µg / mL or more, about 375 µg / mL or more, about 400 µg / mL or more). In some aspects, the solution can include about 400 µg / mL or less of the lysate or extract (e.g., about 375 µg / mL or less, about 350 µg / mL or less, about 325 µg / mL or less, about 300 µg / mL or less, about 275 µg / mL or less, about 250 µg / mL or less, about 225 µg / mL or less, about 200 µg / mL or less, about 190 µg / mL or less, about 180 µg / mL or less, about 170 µg / mL or less, about 160 µg / mL or less, about 150 µg / mL or less, about 140 µg / mL or less, about 130 µg / mL or less, about 120 µg / mL or less, about 110 µg / mL or less, about 100 µg / mL or less, about 90 µg / mL or less, about 80 µg / mL or less, about 70 µg / mL or less, about 60 µg / mL or less, about 50 µg / mL or less, about 45 µg / mL or less, about 40 µg / mL or less, about 35 µg / mL or less, about 30 µg / mL or less, about 25 µg / mL or less, about 20 µg / mL or less, about 15 µg / mL or less, about 10 µg / mL or less, about 5 µg / mL or less).

[0046] It is considered that the solution can include an amount of the lysate or extract ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the solution can include from about 5 µg / mL to about 400 µg / mL of the lysate or extract (e.g., from about 10 µg / mL to about 375 µg / mL, from about 15 µg / mL to about 350 µg / mL, from about 20 µg / mL to about 325 µg / mL, from about 25 µg / mL to about 300 µg / mL, from about 30 µg / mL to about 275 µg / mL, from about 35 µg / mL to about 250 µg / mL, from about 40 µg / mL to about 225 µg / mL, from about 45 µg / mL to about 200 µg / mL, from about 50 µg / mL to about 190 µg / mL, from about 60 µg / mL to about 180 µg / mL, from about 70 µg / mL to about 170 µg / mL, from about 80 µg / mL to 160 µg / mL, from about 90 µg / mL to about 150 µg / mL, from about 100 µg / mL to about 140 µg / mL, from about 110 µg / mL to about 130 µg / mL, from about 5 µg / mL to about 120 µg / mL, from about 10 µg / mL to about 110 µg / mL, from about 15 µg / mL to about 100 µg / mL, from about 20 µg / mL to about 90 µg / mL, from about 25 µg / mL to about 80 µg / mL, from about 30 µg / mL to about 70 µg / mL, from about 35 µg / mL to about 60 µg / mL, from about 40 µg / mL to about 50 µg / mL, from about 120 µg / mL to about 400 µg / mL, from about 120 µg / mL to about 375 µg / mL, from about 130 µg / mL to about 350 µg / mL, from about 140 µg / mL to about 325 µg / mL, from about 150 µg / mL to about 300 µg / mL, from about 160 µg / mL to about 275 µg / mL, from about 170 µg / mL to about 250 µg / mL, from about 180 µg / mL to about 225 µg / mL, from about 190 µg / mL to about 200 µg / mL). ^Attorney Docket No.11001-206WO1

[0047] In some aspects, the lysate or extract can include homogenized cellular components of Enterococcus Avium strain HL-95 (SEQ ID NO: 2).

[0048] In some aspects, the hydrogel can further include betadine containing 10% povidone-iodine. The hydrogel can include betadine in any suitable amount. For example, in some aspects, the hydrogel can include about 0.01 wt% betadine or more (e.g., about 0.02 wt% or more, about 0.03 wt% or more, about 0.04 wt% or more, about 0.05 wt% or more, about 0.1 wt% or more, about 0.15 wt% or more, about 0.2 wt% or more, about 0.25 wt% or more, about 0.5 wt% or more, about 0.75 wt% or more, about 1 wt% or more, about 1.5 wt% or more, about 2 wt% or more, about 2.5 wt% or more, about 3 wt% or more, about 3.5 wt% or more, about 4 wt% or more, about 4.5 wt% or more, about 5 wt% or more, about 6 wt% or more, about 7 wt% or more, about 8 wt% or more, about 9 wt% or more, about 10 wt% or more). In some aspects, the hydrogel can include about 10 wt% betadine or less (e.g., about 9 wt% or less, about 8 wt% or less, about 7 wt% or less, about 6 wt% or less, about 5 wt% or less, about 4.5 wt% or less, about 4 wt% or less, about 3.5 wt% or less, about 3 wt% or less, about 2.5 wt% or less, about 2 wt% or less, about 1.5 wt% or less, about 1 wt% or less, about 0.75 wt% or less, about 0.5 wt% or less, about 0.25 wt% or less, about 0.2 wt% or less, about 0.15 wt% or less, about 0.1 wt% or less, about 0.05 wt% or less, about 0.04 wt% or less, about 0.03 wt% or less, about 0.02 wt% or less, about 0.01 wt% or less).

[0049] It is considered that the hydrogel can include an amount of betadine ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the hydrogel can include from about 0.01 wt% to about 10 wt% betadine (e.g., from about 0.02 wt% to about 9 wt%, from about 0.03 wt% to about 8 wt%, from about 0.04 wt% to about 7 wt%, from about 0.05 wt% to about 6 wt%, from about 0.1 wt% to about 5 wt%, from about 0.15 wt% to about 4.5 wt%, from about 0.2 wt% to about 4 wt%, from about 0.25 wt% to about 3.5 wt%, from about 0.5 wt% to about 3 wt%, from about 1 wt% to about 2.5 wt%, from about 1.5 wt% to about 2 wt%, from about 0.01 wt% to about 2 wt%, from about 0.02 wt% to about 1.5 wt%, from about 0.03 wt% to about 1 wt%, from about 0.04 wt% to about 0.75 wt%, from about 0.05 wt% to about 0.5 wt%, from about 0.1 wt% to about 0.25 wt%, from about 0.15 wt% to about 0.2 wt%, from about 1.5 wt% to about 10 wt%, from about 2 wt% to about 9 wt%, from about 2.5 wt% to about 8 wt%, from about 3 wt% to about 7 wt%, from about 3.5 wt% to about 6 wt%, from about 4 wt% to about 5 wt%). METHODS

[0050] In an aspect, provided is a method of enhancing or promoting wound healing, the method including topically administering a lysate or extract from Lactobacillus ^Attorney Docket No.11001-206WO1 Argentoratensis strain HL-198 (SEQ ID NO: 1) to wounded, damaged, or inflamed skin. In some aspects, the lysate or extract can include homogenized cellular components of Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1). In some aspects, the wounded, damaged, or inflamed skin can be re-epithelized by about 85% or more after 24 hours (e.g., about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100%).

[0051] In another aspect, provided is a method of enhancing or promoting wound healing, the method including topically administering a lysate or extract from Enterococcus Avium strain HL-95 (SEQ ID NO: 2) to wounded, damaged, or inflamed skin. In some aspects, the lysate or extract can include homogenized cellular components of Enterococcus Avium strain HL-95 (SEQ ID NO: 2). In some aspects, the wounded, damaged, or inflamed skin can be re- epithelized by about 70% or more (about 71% or more, about 72% or more, about 73% or more, about 74% or more, about 75% or more, about 76% or more, about 77% or more, about 78% or more, about 79% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 100% or more) after 24 hours.

[0052] In some aspects, the lysate or extract can be administered at least once a day (e.g., 1, 2, 3, 4, 5, 6, or more times per day) for 14 days or less (e.g., 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, 1 day). In some aspects, the lysate or extract can be administered at least once a day (e.g., 1, 2, 3, 4, 5, 6, or more times per day) for 14 days or more (e.g., 15 days or more, 16 days or more, 17 days or more, 18 days or more, 19 days or more, 20 days or more, 21 days or more, 28 days or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more).

[0053] In yet another aspect, provided is a method of skin protection, the method including topically administering a lysate or extract from Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1) to skin before exposure to a wounding agent. In some aspects, the lysate or extract can include homogenized cellular components of Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1). ^Attorney Docket No.11001-206WO1

[0054] In yet still another aspect, provided is a method of skin protection, the method including topically administering a lysate or extract from Enterococcus Avium strain HL-95 (SEQ ID NO: 2) to skin before exposure to a wounding agent. In some aspects, the lysate or extract can include homogenized cellular components of Enterococcus Avium strain HL-95 (SEQ ID NO: 2).

[0055] In some aspects, the wounding agent can be UV radiation.

[0056] In some aspects, cell viability of the skin can be decreased by about 45% or less after exposure to the wounding agent (e.g., about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less).

[0057] In some aspects, the lysate or extract can be administered 1 minute or more before exposure to the wounding agent (e.g., 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more, 1.5 hours or more, 2 hours or more, 4 hours or more, 8 hours or more, 12 hours or more, 24 hours or more, 48 hours or more, 72 hours or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more). In some aspects, the lysate or extract can be administered 14 days or less before exposure to the wounding agent (e.g., 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 72 hours or less, 48 hours or less, 24 hours or less, 12 hours or less, 8 hours or less, 4 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less).

[0058] It is considered that the lysate or extract can be administered any amount of time before exposure to the wounding agent ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the lysate or extract can be administered from 1 minute to 14 days before exposure to the wounding agent (e.g., from 2 minutes to 13 days, from 3 minutes to 12 days, from 4 minutes to 11 days, from 5 minutes to 10 days, from 10 minutes to 9 days, from 15 minutes to 8 days, from 30 minutes to 7 days, from 45 minutes to 6 days, from 1 hour to 5 days, from 1.5 hours to 4 days, from 2 hours to 72 hours, from 4 hours to 48 hours, from 8 hours to 24 hours, from 1 minute to 24 hours, from 2 minutes to 12 hours, from 3 minutes to 8 hours, from 4 minutes to 4 hours, from ^Attorney Docket No.11001-206WO1 5 minutes to 2 hours, from 10 minutes to 1.5 hours, from 15 minutes to 1 hour, from 30 minutes to 45 minutes, from 24 hours to 14 days, from 48 hours to 13 days, from 72 hours to 12 days, from 4 days to 11 days, from 5 days to 10 days, from 6 days to 9 days, from 7 days to 8 days)

[0059] In some aspects, any of the disclosed methods can further include co-administration of betadine containing 10% povidone-iodine to the skin.

[0060] In some aspects, the lysate or extract used in any of the disclosed methods can be encapsulated in a hydrogel. In some aspects, the hydrogel can include hydroxypropyl cellulose. In some aspects, the hydrogel can be crosslinked with citric acid.

[0061] In some aspects, the hydrogel can be prepared from a solution including hydrogel precursor and the lysate or extract (for example, the solution can be crosslinked or cured to form the hydrogel). In some aspects, the solution can include about 5 µg / mL or more of the lysate or extract (e.g., about 10 µg / mL or more, about 15 µg / mL or more, about 20 µg / mL or more, about 25 µg / mL or more, about 30 µg / mL or more, about 35 µg / mL or more, about 40 µg / mL or more, about 45 µg / mL or more, about 50 µg / mL or more, about 60 µg / mL or more, about 70 µg / mL or more, about 80 µg / mL or more, about 90 µg / mL or more, about 100 µg / mL or more, about 110 µg / mL or more, about 120 µg / mL or more, about 130 µg / mL or more, about 140 µg / mL or more, about 150 µg / mL or more, about 160 µg / mL or more, about 170 µg / mL or more, about 180 µg / mL or more, about 190 µg / mL or more, about 200 µg / mL or more, about 225 µg / mL or more, about 250 µg / mL or more, about 275 µg / mL or more, about 300 µg / mL or more, about 325 µg / mL or more, about 350 µg / mL or more, about 375 µg / mL or more, about 400 µg / mL or more). In some aspects, the solution can include about 400 µg / mL or less of the lysate or extract (e.g., about 375 µg / mL or less, about 350 µg / mL or less, about 325 µg / mL or less, about 300 µg / mL or less, about 275 µg / mL or less, about 250 µg / mL or less, about 225 µg / mL or less, about 200 µg / mL or less, about 190 µg / mL or less, about 180 µg / mL or less, about 170 µg / mL or less, about 160 µg / mL or less, about 150 µg / mL or less, about 140 µg / mL or less, about 130 µg / mL or less, about 120 µg / mL or less, about 110 µg / mL or less, about 100 µg / mL or less, about 90 µg / mL or less, about 80 µg / mL or less, about 70 µg / mL or less, about 60 µg / mL or less, about 50 µg / mL or less, about 45 µg / mL or less, about 40 µg / mL or less, about 35 µg / mL or less, about 30 µg / mL or less, about 25 µg / mL or less, about 20 µg / mL or less, about 15 µg / mL or less, about 10 µg / mL or less, about 5 µg / mL or less).

[0062] It is considered that the solution can include an amount of the lysate or extract ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the solution can include from about 5 µg / mL to about 400 µg / mL of the lysate or extract (e.g., from about 10 µg / mL to about 375 µg / mL, ^Attorney Docket No.11001-206WO1 from about 15 µg / mL to about 350 µg / mL, from about 20 µg / mL to about 325 µg / mL, from about 25 µg / mL to about 300 µg / mL, from about 30 µg / mL to about 275 µg / mL, from about 35 µg / mL to about 250 µg / mL, from about 40 µg / mL to about 225 µg / mL, from about 45 µg / mL to about 200 µg / mL, from about 50 µg / mL to about 190 µg / mL, from about 60 µg / mL to about 180 µg / mL, from about 70 µg / mL to about 170 µg / mL, from about 80 µg / mL to 160 µg / mL, from about 90 µg / mL to about 150 µg / mL, from about 100 µg / mL to about 140 µg / mL, from about 110 µg / mL to about 130 µg / mL, from about 5 µg / mL to about 120 µg / mL, from about 10 µg / mL to about 110 µg / mL, from about 15 µg / mL to about 100 µg / mL, from about 20 µg / mL to about 90 µg / mL, from about 25 µg / mL to about 80 µg / mL, from about 30 µg / mL to about 70 µg / mL, from about 35 µg / mL to about 60 µg / mL, from about 40 µg / mL to about 50 µg / mL, from about 120 µg / mL to about 400 µg / mL, from about 120 µg / mL to about 375 µg / mL, from about 130 µg / mL to about 350 µg / mL, from about 140 µg / mL to about 325 µg / mL, from about 150 µg / mL to about 300 µg / mL, from about 160 µg / mL to about 275 µg / mL, from about 170 µg / mL to about 250 µg / mL, from about 180 µg / mL to about 225 µg / mL, from about 190 µg / mL to about 200 µg / mL).

[0063] In some aspects, the hydrogel can include any of the disclosed hydrogels. EXAMPLES Example 1: Bacterial strains for wound healing and skin protection

[0064] This study focuses on the identification and evaluation of two bacterial strains, HL- 95 and HL-198, as postbiotic agents with the potential for promoting wound healing.

[0065] Postbiotics, which are metabolites or cellular components derived from probiotics, have been increasingly studied for their potential therapeutic effects, including modulation of the immune response and promotion of tissue regeneration [5], [6]. Unlike live probiotics, postbiotics do not carry the risk of causing infections or unwanted bacterial growth, making them safer and more practical for therapeutic applications such as wound healing [6], [7]. The bioactive compounds produced by beneficial bacteria can exert anti-inflammatory, antimicrobial, and tissue-regenerative effects, thus influencing multiple stages of the wound healing process [8]. Several bacterial strains have been investigated for their potential to enhance wound healing, with some showing capabilities to modulate the inflammatory phase and promote epithelial regeneration and collagen synthesis [9].

[0066] In this context, screening bacterial strains for their postbiotic effects on wound healing is a crucial step in identifying candidates with the highest therapeutic potential. A study was conducted which screened 167 different bacterial strains and evaluated their efficacy using the scratch assay on 3T3-L1 fibroblast cells. The scratch assay is a widely used in vitro ^Attorney Docket No.11001-206WO1 technique that mimics wound closure by creating a gap (or "scratch") in a confluent monolayer of cells and observing the rate of cell migration and proliferation into the wound space. This method provides valuable insights into the wound healing potential of various treatments by assessing the re-epithelialization process, a critical aspect of tissue repair

[0010] . After the initial screening of 167 bacterial strains using the scratch assay on 3T3-L1 cells, 30 strains showed notable wound healing potential based on their ability to accelerate fibroblast migration and wound closure. These 30 strains were then further evaluated using the scratch assay on BJ human fibroblast cells, a more relevant model for studying wound healing in humans. From this secondary screening, the study selected seven bacterial strains that demonstrated superior wound healing activity in terms of both speed and extent of wound closure. Among these, strains HL-95 and HL-198 were found to be the most effective. The selected bacterial strains, HL-95 and HL-198 were then subjected to further investigation to assess their effects on various key parameters associated with wound healing. The scratch assay was used to measure the extent of re-epithelialization, which is critical for restoring the skin barrier

[0011] . Accelerated re-epithelialization indicates the potential to enhance keratinocyte migration and proliferation, processes essential for wound closure

[0012] . The inflammatory phase of wound healing is characterized by the release of pro-inflammatory cytokines such as TNF-^, IL-6, and IL-1^, which are necessary for clearing debris and preventing infection

[0013] . However, prolonged or excessive inflammation can delay healing. The study measured the expression of matrix metalloproteinase-9 (MMP-9), which is an enzyme that plays a crucial role in tissue remodeling, inflammation, and wound healing. It is involved in the degradation of the extracellular matrix (ECM) and is regulated during inflammatory responses

[0014] . The modulation of these inflammatory markers is crucial for transitioning from the inflammatory to the proliferative phase of healing

[0015] . Further, fibrillin is a glycoprotein that is integral to the formation of elastic fibers, which provide resilience and strength to the skin

[0016] . Collagen, on the other hand, is the primary structural protein in the extracellular matrix and is essential for tissue repair and scar formation

[0016] . To assess the quality of the newly formed tissue, the study measured fibrillin expression using immunofluorescence staining and collagen content using the hydroxyproline assay. Measuring these parameters would suggest that bacterial extract not only promotes faster wound closure but also enhances the structural integrity of the repaired tissue

[0017] .

[0067] To validate the in vitro findings and explore the therapeutic potential of HL-95 and HL-198 in a more complex biological system, an in vivo wound healing study was conducted using a mouse model. Full-thickness wounds were created on the dorsal skin of mice using a ^Attorney Docket No.11001-206WO1 punch biopsy, and the wounds were treated with hydrogels containing postbiotic extracts from either HL-95 or HL-198. Wound closure was monitored over 14 days, with measurements taken on day 0, day 7, and day 14. The hydrogel formulation provided a sustained release of the bacterial extracts, ensuring continuous exposure of the wound site to the bioactive compounds. Histological analysis using Hematoxylin and Eosin (H&E) staining was performed to evaluate epithelialization, while collagen content was measured through the hydroxyproline assay. Additionally, MMP-9 and fibrillin were analyzed through immunofluorescence assays. Materials and Methods

[0068] Bacterial Strain Collection and Culture: Following the previously reported procedure

[0018] , the strains of Lactobacillus Argentoratensis (HL-198) and Enterococcus Avium (HL-95) were isolated on De Man, Rogosa, Sharpe (MRS) agar and then moved to a liquid medium (MRS). The cultures were cultured for 12–24 hours at 37°C after inoculating 1 mL of bacterial suspension into 9 mL of MRS to prepare the stocks and kept at −80°C in 20% glycerol.

[0069] Cell Lines and Culture Conditions: 3T3-L1 Mouse fibroblasts, plays a pivotal role in wound healing studies due to its ability to produce collagen and other extracellular matrix components essential for tissue repair. BJ Human fibroblasts cells were cultured in DMEM and EMEM respectively, with 10% FBS and 1% penicillin / streptomycin. Both 3T3- L1 and BJ cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.3T3- L1 between passages 6 and 8 were used for experiments and were collected using a trypsin- EDTA solution.3T3-L1 Cells.

[0070] In Vitro Scratch Assay on 3T3-L1 and BJ Cells: An in vitro scratch assay was performed on 3T3-L1 cells to evaluate the wound healing potential of seven most promising strains such as HL-33, HL-73, HL-94, HL-95, HL-158, HL-198, and HL-207. Further, the Secondary Screening on BJ Fibroblasts cells. Two strains showing the highest wound closure rates such as HL-95 and HL-198 in 3T3-L1 assays were selected for further testing on BJ fibroblasts. The scratch assay was repeated with BJ cells, and wound closure was assessed at 0, 6, 12, and 24 hours. Briefly, the BJ were seeded at a density of 5^×^105cells / well in 24-well cell culture plates. Cells were treated with HL-95 and HL-198 respectively. 100% confluent cell monolayers were scratched across the well with a 200 µL sterile micropipette tip. Debris and dislodged cells were then removed from the wells using Dulbecco's phosphate-buffered saline (Sigma). An Olympus IX73 microscope with a digital camera was used to obtain pictures of the wound closure at 0, 6, 12, and 24 hours. ImageJ software was used to calculate re- epithelization rates as an average percentage of the wound area. ^Attorney Docket No.11001-206WO1 Re-epithelized area (%) = (Wound area on day zero − Wound area on last day ) / (Wound area on day zero) × 100

[0019]

[0071] Protein extraction: The microbial strains were grown in MRS overnight and then centrifuged at 900g for 5 min. then the supernatant discarded and the pellets were re-suspended in 500 ^L PBS. After that the cells were subjected to three ultrasonication (30 s each with 1 min interval on ice) using Sonicator (Qsonica, LLC) with an amplitude of 25%. Remaining cell debris was removed through high-speed centrifugation at 16,000g, 4°C for 10 min. Then the protein concentration was determined.

[0072] Hydrogel Preparation: Bacterial Extract Derived hydrogel (BEDELs) preparation was made with hydroxypropyl cellulose (HPC) using the casting technique. In order to create a homogenous solution, 2.0 g HPC was added to 100 mL of distilled water and constantly agitated at 60°C for one hour. The crosslinking procedure was then finished by adding 0.5% citric acid and stirring continuously for two hours. Subsequently, a homogenous solution was obtained by mixing bacterial extract of HL-95 and HL-198 at a concentration of 100 ug / ml with the solution and agitating for 30 minutes. Lastly, 15 mL of the mixes were put in a Petri dish and left to dry at 50°C for the entire night. The hydrogel were then kept in a desiccator until needed

[0020] .

[0073] Animal Studies: Wild-type C57BL / 6J mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). They were acclimatized for two weeks in the vivarium with controlled temperature of 22°C and a 12-hour light-dark cycle before experiments began. Three to five (maximum five) littermates were housed in each cage, with males and females housed separately, and were fed with normal chow and tap water. All the tests and procedures that involved animals were performed as per Animal Research: Reporting of In vivo Experiments (ARRIVE) guidelines, and all the experimental protocols were approved by Institutional Animal Care and Use Committee (IACUC) of the University of South Florida, Tampa, Florida. All methods were performed in accordance with the relevant guidelines and regulations.

[0074] In Vivo Wound Healing Model:

[0075] Experimental groups: To determine the role of different formulations of Bacterial Extract Derived hydrogel (BEDELs) derived from selected bacteria strains on wound healing, n=40 wild-type C57BL / 6J mice aged 4-6 weeks old (n=10) was randomly divided into the following four groups: Control, HL-95, HL-198 and Betadine. ^Attorney Docket No.11001-206WO1

[0076] Intervention and Procedure Schedules: The mice were divided into four main groups, and each group contained 10 randomly selected mice. Before wounding surgery, each mouse was injected subcutaneously with meloxicam 5-10 mg / kg of body weight (0.3 ml) and once daily for 48 hours postoperatively up to 5 days and as required later. All the mice anesthetized by isoflurane inhalation and the back of mice were shaved by using hair clipper / shaver. Germicidal scrub Avagard was applied to prepare the dorsum for wounding. A biopsy punch has been used to create a 5^mm diameter circular full-thickness skin wound on the dorsum. The wound is formed in the paravertebral region of the animal’s dorsum. Immediately after the infliction of the wound, the wounded areas were applied with 50 µL of placebo, HL- 95, HL-198 and betadine twice a day, respectively. Before, taking pictures, mice were anesthetized by isoflurane inhalation. Pictures of the wound area were captured on day-0, day- 7 and day-14. On the last day of experiment all the mice were euthanized, and the samples were collected. Half of the wound samples of each mouse were used for histopathological examination and half of them were used in molecular analysis.

[0077] Histological Analysis and Collagen Measurement:

[0078] Hematoxylin and Eosin (H&E) staining: The 25 µm thick sections of OCT- embedded tissue were stained with H&E (Cat#ab245880, abcam, USA) as per the protocol to evaluate epithelialization. The images were captured using Olympus on 10× magnification. Thickness of epidermis measured using FIJI software (NIH).

[0079] Immunofluorescence: To perform immunofluorescence of MMP-9 and fibrillin, the 25 µm skin section slides were kept in the sodium citrate buffer for 30 min at 700C (n=5). After this, Phosphate-buffered saline (PBS) was used to wash them three times to prevent masking. After applying methanol to the sections, they were incubated in 5% blocking buffer for one hour

[0021] , and then incubated for an additional night at 4°C with one or another of the following primary antibodies: anti-MMP-9 (rabbit) antibody (Cat# 13667, CST, USA) or anti-fibrillin (rabbit) antibody (Cat# PA5-99225, Invitrogen, USA). The slices were treated with the alexa fluorTM594 (Cat#A11007, Invitrogen, USA) and alexa fluorTM488 (Cat#A11034, Invitrogen, USA) relevant secondary antibodies for 1 hour at room temperature following PBS washing. Then the sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Fluorescence photomicrography was done using an Olympus 1200 confocal microscope set at 10x and 20x magnification.

[0080] mRNA expression-qRT-PCR: Total RNA from tissues was extracted with RNeasy and transcribed to cDNA with a High-capacity cDNA Reverse Transcription Kit as described earlier

[0022] ,

[0023] . Real-time PCR was performed using Real Time PCR system (Cat#A28132 ^Attorney Docket No.11001-206WO1 Quant Studia 3, USA) to measure the expression of MMP-9, Il-6, Il-1^, and Tnf-^ (n=5).The study employed 18S rRNA as an internal control (primer details are presented in TABLE 1). Using the ^^Ct method, fold changes were calculated for each quantitative PCR (qPCR) analysis, which was conducted in duplicate as technical replicate while each individual sample as biological replicate. TABLE 1. Forward and reverse sequence of primers used in the experiment during RTqPCR.

[0081] Statistical analyses: One-way ANOVAs were used to analyze the datasets using GraphPad Prism v9.0. Two-way ANOVA followed by Tukey’s multiple comparison test were applied for the analysis of wound healing for both in vitro and in vivo. All the data are presented as mean and standard deviation and *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 were considered statistically significant.

[0082] Unpaired Student's t-test and one-way ANOVA were used to analyze the datasets using GraphPad Prism v9.0. An unpaired two-tailed t-test was used to compare the alpha diversity indices and bacterial abundance between the two groups. The bacterial beta diversity was presented in principal component analysis (PCA) format and analysed using packages ‘ggplot2’, ‘devtools’ and ‘ggbiplot’ in R programming V.4.2.0

[0034] . Moreover, bacterial taxonomy between control and probiotics groups were analyzed using linear discriminant analysis (LDA) effect size (LEfSe)

[0035] . Microbiome Multivariate Association with Linear model 2 (MaAsLiN2) analysis was conducted to determine the association of significant bacterial taxa between control and probiotics groups with various phenotypic and genotypic ^Attorney Docket No.11001-206WO1 functional

[0034] ,

[0036] . Two-way ANOVA followed by Tukey’s multiple comparison test were applied for the sex-based comparison. All the data are presented as mean and standard deviation and *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 were considered statistically significant. Results

[0083] Screening of HL-95 and HL-198 among 240 bacterial strains: The study began by screening 240 proposed / putatively identified probiotic candidates (see in FIG. 1A). The bacterial lysates were prepared using bead beating and ultrasonication, followed by protein content estimation and normalization. Strains with extremely low protein content were excluded from the study based on a stringent quality control (QC) process. As a result, 167 strains were selected for further investigation. Each batch of lysate was normalized to ensure that all wells in the scratch assay plate received a standardized amount of lysate, regardless of the strain. As a result, 167 strains were selected for further investigation. Each batch of lysate was normalized to ensure that all wells in the scratch assay plate received a standardized amount of lysate, regardless of the strain. The scratch assay was first performed on the 3T3 L1 cell line, which identified 30 strains with the best wound closure abilities (represented in FIG. 1B). These top 30 strains were then tested on the BJ cell line, a human skin cell line crucial to wound healing. After further analysis, 7 strains demonstrating wound closure capabilities (see in FIG.1C), however, HL-95 and HL-198 were found to be more effective and were selected for further in vitro and in vivo study. The 3T3 L1 cell line, derived from mouse fibroblasts, plays a pivotal role in wound healing studies due to its ability to produce collagen and other extracellular matrix components essential for tissue repair. These cells are commonly used in in vitro scratch assays to study cellular migration, allowing researchers to evaluate the effectiveness of compounds, such as postbiotic lysates, in promoting wound closure by enhancing cell movement. On the other hand, the BJ cell line, which originates from human foreskin fibroblasts, closely mimics human skin fibroblast behavior. This makes it especially valuable for modeling human-specific wound healing processes, such as collagen synthesis, proliferation, and migration. Testing postbiotic effects on BJ cells offers clinically relevant insights into how these bacterial lysates might influence wound healing in humans, providing a more comprehensive understanding of postbiotic efficacy.

[0084] HL-198 potentiates time-dependent re-epithelialization by reducing inflammatory markers and ROS production in invitro wound model: FIGS.2A-2B show the time-dependent re-epithelialization rates for control, HL-95, and HL-198 treatments over a 24-hour period, with re-epithelialization measured at 0, 6, 12, and 24 hours. At 6 hours, both ^Attorney Docket No.11001-206WO1 HL-95 and HL-198 treatments showed significantly increased re-epithelialization compared to the control group. HL-198 resulted in the highest rate of re-epithelialization, reaching approximately 35%, while HL-95 showed a rate of around 30%, and the control remained below 20%. At 12 hours, the differences became more pronounced, with HL-198 achieving a re-epithelialization rate of around 65%, while HL-95 followed closely with 55%. The control group showed a smaller increase, reaching approximately 40%.

[0085] At 24 hours, HL-198 continued to show superior results with nearly 90% re- epithelialization, significantly outperforming both HL-95 (approximately 75%) and the control group (around 55%). Statistical analysis indicated that HL-198's effect was highly significant (p < 0.01) compared to the control, and HL-95 also demonstrated a significant difference (p < 0.05) compared to the control. Therefore, HL-198 exhibited the most potent effect in enhancing re-epithelialization over the 24-hour period, followed by HL-95, both of which significantly outperformed the control treatment.

[0086] FIG. 2C above shows reactive oxygen species (ROS) generation in cells treated with HL-95 and HL-198, compared to the control group. ROS production was detected using H2DCFDA (green fluorescence), and cell nuclei were stained with DAPI (blue fluorescence). The merged images (DAPI / H2DCFDA) indicate the colocalization of ROS production and cell nuclei.

[0087] In the control group, there is high-intensity green fluorescence indicating elevated ROS levels. This suggests that untreated cells experienced significant oxidative stress, as shown by the abundant H2DCFDA signal. In cells treated with HL-95, a moderate reduction in ROS levels is observed. The green fluorescence is diminished compared to the control group, indicating a decrease in oxidative stress within these cells. Cells treated with HL-198 exhibited the most significant reduction in ROS generation. The green fluorescence is notably weaker compared to both the control and HL-95 groups, suggesting that HL-198 effectively reduced oxidative stress in the treated cells. Moreover, there was a significant reduction in inflammatory markers such as MMP-9, Tnf-^, Il-6 and Il-1^ expression following HL-198 treatment however HL-95 ameliorated only Il-6 (FIGS. 2D-2H). Additionally, treatment with HL -198 increased the expression of fibrillin-1 protein in BJ cells (FIG.2I).

[0088] HL-198 facilitates wound closure by potentiating fibrillin-1 and reducing inflammatory markers: FIG.3A represented the wound sites over a time course show visible changes in wound closure across different groups or treatments (as indicated in the top panel of images). The initial wound exhibits a circular morphology with a visible opening (Day 0). As time progresses, some wounds exhibit scabbing and partial closure, while others appear to ^Attorney Docket No.11001-206WO1 close more effectively, as seen in later time points. In some cases, the wounds are covered with a layer of tissue indicative of advanced healing, while others show signs of incomplete closure or scab formation. FIG. 3B showed wound closure percentage over time, with HL-198 and betadine demonstrating significantly faster healing (as indicated by statistical markers).

[0089] Further, FIG. 3C showed immunofluorescent staining for MMP-9 (green) and DAPI (blue). MMP-9, an enzyme involved in extracellular matrix remodeling during wound healing, shows increased expression at the wound site over time, particularly in regions where wound closure is incomplete. The merged images (DAPI / MMP-9) highlight co-localization of nuclei (stained with DAPI) and areas of MMP-9 expression, as indicated by the red arrows. The areas of strong MMP-9 expression appear more abundant at control, with a reduction at HL-95, HL-198 and betadine correlating with the wound healing process. The data suggests that MMP-9 plays a critical role in the early phases of wound healing, contributing to matrix remodeling. The wound healing rates vary between groups, with certain treatments potentially promoting faster wound closure and reduced MMP-9 activity in the later stages of healing. Moreover, there is a significant decrease in the expression of inflammatory makers such Tnf- ^, Il-6 and Il-1^ following HL-198 and betadine topical application for 14 days.

[0090] HL-198 facilitates epithelization by potentiating collagen formation: The histological analysis using hematoxylin and eosin (H&E) staining reveals differences in the epidermal structure across the control and experimental groups (HL-95, HL-198, and Betadine).

[0091] The control group exhibits thinner epidermal layers with relatively sparse dermal- epidermal junctions. Hair follicles are less developed, with limited thickening of the epidermis. Treatment with HL-95 shows moderate epidermal thickening compared to the control. There is an increase in the density of hair follicles and connective tissue beneath the epidermis, indicating a possible stimulatory effect on tissue regeneration and repair. HL-198 treatment exhibits the most pronounced epidermal thickening, with well-developed and denser hair follicles. The overall structure shows a significant increase in epidermal and dermal thickness, suggesting more effective regeneration and healing in comparison to both the control and HL- 95 groups. The Betadine group also shows significant epidermal thickening, with well-defined hair follicles. However, the organization of the tissue appears slightly different from the HL- 198 group, with potentially more fibrous tissue underneath the epidermis (see FIGS. 3A-3B). FIGS.3C-3D show hydroxyproline content, and Masson’s trichrome histology an indicator of collagen deposition and connective tissue regeneration, reveals significant differences between the groups. The control group displays the lowest levels of hydroxyproline, indicating minimal ^Attorney Docket No.11001-206WO1 collagen deposition and tissue repair in comparison to the treated groups. The HL-198 and betadine shows a significantly higher hydroxyproline concentration compared to both the control and HL-95 groups, reflecting enhanced connective tissue formation and a more robust healing response.

[0092] Therefore, HL-198 treatments improved wound healing and collagen deposition in the skin compared to the control, with showing more significant improvements in epidermal thickness and collagen synthesis demonstrated high efficacy in promoting tissue regeneration, performing similarly or better than betadine in terms of hydroxyproline content, but with slight variations in tissue structure. Based on these findings, HL-198 shows promising potential as a treatment for enhancing wound healing and tissue repair, comparable to standard treatments like Betadine. Discussion

[0093] The present study aimed to explore the wound healing potential of bacterial strains screened from a pool of 167 isolates using a multi-step screening process. By employing in vitro scratch assays on 3T3-L1 and BJ cell lines, the study identified two strains, HL-95 and HL-198, as the most promising candidates for wound healing. These results demonstrate that both HL-95 and HL-198 significantly enhance wound healing parameters such as re- epithelialization, collagen formation, and the modulation of inflammatory markers. Notably, HL-198 proved to be the most effective in reducing inflammatory responses and promoting tissue repair.

[0094] The initial scratch assays performed on 3T3-L1 and BJ cell lines allowed for the high-throughput screening of bacterial strains. Among them, time-dependent scratch assays revealed that HL-95 and HL-198 were significantly more effective in promoting re- epithelialization. HL-198, in particular, accelerated wound closure in vitro, with nearly 90% re-epithelialization observed at 24 hours compared to 75% for HL-95 and 55% for the control (FIGS. 4A-4C). This rapid re-epithelialization is a critical component of effective wound healing, as epithelial cells must quickly cover the wound surface to prevent infection and restore barrier function

[0010] . In support of these findings previous studies have shown that heat killed bacterial strain or postbiotics promotes epithelial migration and proliferation

[0024] ,

[0025] . Additionally, during in vivo study, the time-dependent healing assays confirmed that HL-198 facilitated faster wound closure, particularly during the early phases of healing (day-7) to day- 14, suggesting that it may influence initial cellular responses critical for re-epithelialization. Re-epithelialization is a crucial process in wound healing as it involves the migration of keratinocytes to cover the wound bed and restore the epidermal barrier. Thus, this finding ^Attorney Docket No.11001-206WO1 implicates the significant improvements in re-epithelialization in both HL-95 and HL-198- treated wounds (confirmed by in vitro and in vivo) compared to the control group, with HL- 198 showing the most pronounced effect. This is consistent with other studies where specific bacterial strains or postbiotics have been shown to enhance keratinocyte migration and proliferation, accelerating wound closure

[0024] ,

[0026] .

[0095] Inflammation is a key component of the wound healing process, but prolonged or excessive inflammation can impede healing and lead to chronic wounds. The study measured the mRNA expreesion of pro-inflammatory cytokines Tnf-^, Il-6, and Il-1^, as well as matrix metalloproteinase-9 (MMP-9), which are known to degrade extracellular matrix components during the wound healing process. Both HL-95 and HL-198 significantly reduced the levels of these inflammatory markers in vitro. HL-198 was particularly effective in downregulating MMP-9, Tnf-^, Il-6 and Il-1^ in both in vitro as well as in vivo indicating its anti-inflammatory properties. Impaired MMP-9 levels are associated with impaired healing in chronic wounds, as it can degrade collagen and other matrix proteins essential for tissue repair27,28. The ability of HL-198 to modulate MMP-9 activity further highlights its potential as a therapeutic agent for promoting wound healing by reducing extracellular matrix degradation. Moreover, collagen synthesis is crucial for the structural integrity of healing tissue, and its deposition is a hallmark of the proliferative phase of wound healing and in the management of scar

[0029] . To assess collagen content, the study performed a hydroxyproline assay, which indicated that HL-198 treatment significantly increased collagen deposition compared to HL-95 and the control group. This finding was further corroborated by H&E staining, which revealed enhanced epithelialization and tissue regeneration in HL-198-treated wounds. Collagen provides the scaffold necessary for new tissue formation and supports the migration of fibroblasts and endothelial cells, both of which are essential for wound closure

[0030] . Furthermore, increased fibrillin production was also observed in HL-198-treated samples, as measured through immunofluorescence assays. Fibrillin is a key structural protein in the extracellular matrix that contributes to tissue elasticity and strength. Its increased presence suggests that HL-198 not only promotes healing but also enhances the quality of the regenerated tissue, leading to a more robust and durable repair. Studies have shown that bioactive molecules derived from probiotics and beneficial bacteria can promote collagen synthesis and fibroblast activation

[0031] , which may explain the observed increase in collagen and fibrillin levels following HL-198 treatment.

[0096] Collagen synthesis is a critical aspect of wound healing, as it provides structural integrity to the newly formed tissue. The study assessed collagen content using the hydroxyproline assay, a standard method for quantifying collagen in tissue samples. Both ^Attorney Docket No.11001-206WO1 bacterial strains enhanced collagen deposition in the wound area compared to the control, but HL-198 led to significantly higher collagen levels

[0032] . Furthermore, immunofluorescence staining revealed that fibrillin, an essential component of the extracellular matrix involved in tissue elasticity and strength, was significantly upregulated in HL-198-treated wounds. This indicates that HL-198 not only accelerates wound closure but also improves the quality of the newly formed tissue, potentially reducing the risk of scar formation.

[0097] Overall, the efficacy of HL-198 in promoting wound healing could be attributed to several factors. Firstly, HL-198 may produce bioactive compounds that stimulate keratinocyte migration and proliferation, thereby enhancing re-epithelialization. Secondly, its potent anti- inflammatory properties, evidenced by the reduction in pro-inflammatory cytokines and MMP- 9, suggest that HL-198 helps to resolve inflammation more rapidly, allowing for a smoother transition to the proliferative phase of healing. Finally, HL-198’s ability to increase collagen and fibrillin deposition points to its role in enhancing the structural remodeling of the wound tissue, which is critical for long-term wound stability and function. Conclusion

[0098] This study demonstrates that HL-198 is highly effective in promoting wound healing in an in vitro as well as in vivo wound model. Its ability to accelerate re- epithelialization, reduce inflammatory markers, and enhance collagen and fibrillin deposition makes it a promising candidate for further development as a wound healing therapeutic. While HL-95 also showed beneficial effects, HL-198 was consistently more effective across all measured parameters. Future studies should focus on elucidating the molecular mechanisms underlying the superior efficacy of HL-198 and exploring its potential for clinical applications.

[0099] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein. Reference List 1 Eming, S. A., Krieg, T. & Davidson, J. M. Inflammation in wound repair: molecular and cellular mechanisms. Journal of Investigative Dermatology 127, 514-525 (2007). 2 Eming, S. A., Martin, P. & Tomic-Canic, M. Wound repair and regeneration: mechanisms, signaling, and translation. Science translational medicine 6, 265sr266-265sr266 (2014). ^Attorney Docket No.11001-206WO1 3 Las Heras, K., Igartua, M., Santos-Vizcaino, E. & Hernandez, R. M. Chronic wounds: Current status, available strategies and emerging therapeutic solutions. Journal of controlled release 328, 532-550 (2020). 4 Nicholas-Haizelden, K., Murphy, B., Hoptroff, M. & Horsburgh, M. J. Bioprospecting the skin microbiome: advances in therapeutics and personal care products. Microorganisms 11, 1899 (2023). 5 Ye^ilyurt, N., Yılmaz, B., A^agündüz, D. & Capasso, R. Involvement of probiotics and postbiotics in the immune system modulation. Biologics 1, 89-110 (2021). 6 Szydłowska, A. & Sionek, B. Probiotics and postbiotics as the functional food components affecting the immune response. Microorganisms 11, 104 (2022). 7 Isaac-Bamgboye, F. J., Mgbechidinma, C. L., Onyeaka, H., Isaac-Bamgboye, I. T. & Chukwugozie, D. C. Exploring the Potential of Postbiotics for Food Safety and Human Health Improvement. Journal of nutrition and metabolism 2024, 1868161 (2024). 8 Wegh, C. A., Geerlings, S. Y., Knol, J., Roeselers, G. & Belzer, C. Postbiotics and their potential applications in early life nutrition and beyond. International journal of molecular sciences 20, 4673 (2019). 9 Lima, W. G., de Brito, J. C. M., Cardoso, V. N. & Fernandes, S. O. A. In-depth characterization of antibacterial activity of melittin against Staphylococcus aureus and use in a model of non-surgical MRSA-infected skin wounds. European Journal of Pharmaceutical Sciences 156, 105592 (2021). 10 Liang, C.-C., Park, A. Y. & Guan, J.-L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nature protocols 2, 329-333 (2007). 11 Tang, F. et al. Bioactive glass promotes the barrier functional behaviors of keratinocytes and improves the Re-epithelialization in wound healing in diabetic rats. Bioactive Materials 6, 3496-3506 (2021). 12 Raja, S. K., Garcia, M. S. & Isseroff, R. R. Wound re-epithelialization: modulating keratinocyte migration in wound healing. Front Biosci 12, 2849-2868 (2007). 13 Nirenjen, S. et al. Exploring the contribution of pro-inflammatory cytokines to impaired wound healing in diabetes. Frontiers in immunology 14, 1216321 (2023). ^Attorney Docket No.11001-206WO1 14 Rayment, E. A., Upton, Z. & Shooter, G. Increased matrix metalloproteinase^9 (MMP^ 9) activity observed in chronic wound fluid is related to the clinical severity of the ulcer. British Journal of Dermatology 158, 951-961 (2008). 15 Wang, Z., Qi, F., Luo, H., Xu, G. & Wang, D. Inflammatory microenvironment of skin wounds. Frontiers in immunology 13, 789274 (2022). 16 Halper, J. & Kjaer, M. Basic components of connective tissues and extracellular matrix: elastin, fibrillin, fibulins, fibrinogen, fibronectin, laminin, tenascins and thrombospondins. Progress in heritable soft connective tissue diseases, 31-47 (2014). 17 Gurtner, G. C., Werner, S., Barrandon, Y. & Longaker, M. T. Wound repair and regeneration. Nature 453, 314-321 (2008). 18 Miller, B., Mainali, R., Nagpal, R. & Yadav, H. A newly developed Synbiotic yogurt prevents diabetes by improving the microbiome–intestine–pancreas Axis. International journal of molecular sciences 22, 1647 (2021). 19 Kim, E. et al. Effect of baicalin on wound healing in a mouse model of pressure ulcers. International journal of molecular sciences 24, 329 (2022). 20 El Fawal, G. F., Abu-Serie, M. M., Hassan, M. A. & Elnouby, M. S. Hydroxyethyl cellulose hydrogel for wound dressing: Fabrication, characterization and in vitro evaluation. International journal of biological macromolecules 111, 649-659 (2018). 21 Prajapati, S. K., Ahmed, S., Rai, V., Gupta, S. C. & Krishnamurthy, S. Suvorexant improves mitochondrial dynamics with the regulation of orexinergic and mTOR activation in rats exhibiting PTSD-like symptoms. Journal of Affective Disorders (2024). 22 Yadav, H., Lee, J.-H., Lloyd, J., Walter, P. & Rane, S. G. Beneficial metabolic effects of a probiotic via butyrate-induced GLP-1 hormone secretion. Journal of biological chemistry 288, 25088-25097 (2013). 23 Wang, S. et al. Lipoteichoic acid from the cell wall of a heat killed Lactobacillus paracasei D3-5 ameliorates aging-related leaky gut, inflammation and improves physical and cognitive functions: from C. elegans to mice. Geroscience 42, 333-352 (2020). 24 Nam, Y., Kim, J., Baek, J. & Kim, W. Improvement of cutaneous wound healing via topical application of heat-killed Lactococcus chungangensis CAU 1447 on diabetic mice. Nutrients 13, 2666 (2021). ^Attorney Docket No.11001-206WO1 25 Rawal, S. & Ali, S. A. Probiotics and postbiotics play a role in maintaining dermal health. Food & Function 14, 3966-3981 (2023). 26 Golkar, N. et al. A novel effective formulation of bioactive compounds for wound healing: preparation, in vivo characterization, and comparison of various postbiotics cold creams in a rat model. Evidence^Based Complementary and Alternative Medicine 2021, 8577116 (2021). 27 Lazaro, J. et al. Elevated levels of matrix metalloproteinases and chronic wound healing: an updated review of clinical evidence. Journal of wound care 25, 277-287 (2016). 28 Sabino, F. & auf dem Keller, U. Matrix metalloproteinases in impaired wound healing. Metalloproteinases In Medicine, 1-8 (2015). 29 Xue, M. & Jackson, C. J. Extracellular matrix reorganization during wound healing and its impact on abnormal scarring. Advances in wound care 4, 119-136 (2015). 30 Mathew-Steiner, S. S., Roy, S. & Sen, C. K. Collagen in wound healing. Bioengineering 8, 63 (2021). 31 Lew, L. C. & Liong, M. T. Bioactives from probiotics for dermal health: functions and benefits. Journal of applied microbiology 114, 1241-1253 (2013). 32 Singh, D., Rai, V. & Agrawal, D. K. Regulation of collagen I and collagen III in tissue injury and regeneration. Cardiology and cardiovascular medicine 7, 5 (2023). 33 Huuskonen, L., Anglenius, H., Ahonen, I. & Tiihonen, K. Effects of Bacterial Lysates and Metabolites on Collagen Homeostasis in TNF-^-Challenged Human Dermal Fibroblasts. Microorganisms 11, 1465 (2023). 34 Mishra, S. P. et al. Multivariate statistical data analysis-principal component analysis (PCA). International Journal of Livestock Research 7, 60-78 (2017). 35 Segata, N. et al. Metagenomic biomarker discovery and explanation. Genome biology 12, 1-18 (2011). 36 Mallick, H. et al. Multivariable association discovery in population-scale meta-omics studies. PLoS computational biology 17, e1009442 (2021). ^Attorney Docket No.11001-206WO1 SEQUENCES SEQ ID NO: 1 (Lactobacillus Argentoratensis strain HL-19816S rRNA) ATGGGAGGGGAGGTCTGGCGAGGCTGCTGAGAGTCTGATGGAGCAACGCCGCGT GAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCT GAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTA CGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGG GCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAA CCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGA ACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAA GGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAAC AGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGA GGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGT ACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTG GAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATAC TATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTG CATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG CAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGG TGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCTCTTATGACC TGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAG TAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTA CATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGTGAATACGTTC CCGGGTCTTGTACACACCGCCCGTCACACCATGAAGAGTTTGTAACACCCAAAGT CGTGGGGTTACTTTTAGGAACCAGCCGCCTAAGGGTGGGAAGATGGATTAGGGT GAAGTCGTAACACAAGGGTAACCCGTTA SEQ ID NO: 2 (Enterococcus Avium strain HL-9516S rRNA) GGTGGCCAGCTTCTGGCATCGCTTCTGAGCAGTCTGACCGAGCACGCCGCGTGAG TGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAG AGTAGAATGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGT GCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCG TAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACC ^Attorney Docket No.11001-206WO1 GGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAAT TCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGG CGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAG GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGG GTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTAC GACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGA GCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTT TGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCA TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCA ACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTG ACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTG GGCTACACACGTGCTACAATGGGAAGTACAACGAGTCGCGAAGTCGCGAGGCTA AGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGTAGCTGCAACTCGCCTACAT GAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGTGAATACGTTCCCG GGCTTGTACACACGCCCGTCACACCACGAGAGTTAGTAACACCCGAAGTCGTGA GTACTTTTGAGCAGCGCTAGGTGGATAGATGATGGGTGAGTGATCAGGGAGCCG CGCACACACCAAACGAGGAGT^

Claims

Attorney Docket No.11001-206WO1 What is claimed is:

1. A hydrogel comprising a lysate or extract from Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1). The hydrogel of claim 1, wherein the hydrogel comprises hydroxypropyl cellulose.

3. The hydrogel of any one of claims 1-2, wherein the hydrogel is crosslinked with citric acid.

4. The hydrogel of any one of claims 1-3, wherein the hydrogel is prepared from a solution comprising hydrogel precursor and from about 5 µg / mL to about 400 µg / mL of the lysate or extract.

5. The hydrogel of any one of claims 1-4, wherein the lysate or extract comprises homogenized cellular components of Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1).

6. The hydrogel of any one of claims 1-5, further comprising betadine containing 10% povidone-iodine.

7. A method of enhancing or promoting wound healing, the method comprising topically administering a lysate or extract from Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1) to wounded, damaged, or inflamed skin.

8. The method of claim 7, wherein the wounded, damaged, or inflamed skin is re- epithelized by about 85% or more after 24 hours.

9. The method of any one of claims 7-8, wherein the lysate or extract is administered at least once a day for 14 days or less.

10. A method of skin protection, the method comprising topically administering a lysate or extract from Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1) to skin before exposure to a wounding agent. ^Attorney Docket No.11001-206WO1 11. The method of claim 10, wherein the wounding agent is UV radiation.

12. The method of any one of claims 10-11, wherein cell viability of the skin is decreased by about 45% or less after exposure to the wounding agent.

13. The method of any one of claims 10-12, wherein the lysate or extract is administered from 1 minute to 14 days before exposure to the wounding agent.

14. The method of any one of claims 7-13, further comprising co-administration of betadine containing 10% povidone-iodine to the skin.

15. The method of any one of claims 7-14, wherein the lysate or extract comprises homogenized cellular components of Lactobacillus Argentoratensis strain HL-198 (SEQ ID NO: 1).

16. The method of any one of claims 7-15, wherein the lysate or extract is encapsulated in a hydrogel.

17. The method of claim 16, wherein the hydrogel comprises hydroxypropyl cellulose.

18. The method of any one of claims 16-17, wherein the hydrogel is crosslinked with citric acid.

19. The method of any one of claims 16-18, wherein the hydrogel is prepared from a solution comprising hydrogel precursor and from about 5 µg / mL to about 400 µg / mL of the lysate or extract.

20. A hydrogel comprising a lysate or extract from Enterococcus Avium strain HL-95 (SEQ ID NO: 2).

21. The hydrogel of claim 20, wherein the hydrogel comprises hydroxypropyl cellulose. ^Attorney Docket No.11001-206WO1 22. The hydrogel of any one of claims 20-21, wherein the hydrogel is crosslinked with citric acid.

23. The hydrogel of any one of claims 20-22, wherein the hydrogel is prepared from a solution comprising hydrogel precursor and from about 5 µg / mL to about 400 µg / mL of the lysate or extract.

24. The hydrogel of any one of claims 20-23, wherein the lysate or extract comprises homogenized cellular components of Enterococcus Avium strain HL-95 (SEQ ID NO: 2).

25. The hydrogel of any one of claims 20-24, further comprising betadine containing 10% povidone-iodine.

26. A method of enhancing or promoting wound healing, the method comprising topically administering a lysate or extract from Enterococcus Avium strain HL-95 (SEQ ID NO: 2) to wounded, damaged, or inflamed skin.

27. The method of claim 26, wherein the wounded, damaged, or inflamed skin is re- epithelized by about 70% or more after 24 hours.

28. The method of any one of claims 26-27, wherein the lysate or extract is administered at least once a day for up to about 14 days.

29. A method of skin protection, the method comprising topically administering a lysate or extract from Enterococcus Avium strain HL-95 (SEQ ID NO: 2) to skin before exposure to a wounding agent.

30. The method of claim 29, wherein the wounding agent is UV radiation. The method of any one of claims 29-30, wherein cell viability of the skin is decreased by about 45% or less after exposure to the wounding agent.

32. The method of any one of claims 29-31, wherein the lysate or extract is administered from 1 minute to 14 days before exposure to the wounding agent. ^Attorney Docket No.11001-206WO1 33. The method of any one of claims 26-32, further comprising co-administration of betadine containing 10% povidone-iodine to the skin.

34. The method of any one of claims 26-33, wherein the lysate or extract comprises homogenized cellular components of Enterococcus Avium strain HL-95 (SEQ ID NO: 2).

35. The method of any one of claims 26-34, wherein the lysate or extract is encapsulated in a hydrogel.

36. The method of claim 35, wherein the hydrogel comprises hydroxypropyl cellulose.

37. The method of any one of claims 35-36, wherein the hydrogel is crosslinked with citric acid.

38. The method of any one of claims 35-37, wherein the hydrogel is prepared from a solution comprising hydrogel precursor and from about 5 µg / mL to about 400 µg / mL of the lysate or extract. ^

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