Probiotic compositions comprising lactic acid bacteria and methods for producing and using same

Abstract

Probiotic compositions are disclosed that comprise at least one therapeutic bacterium or a spore thereof, wherein the at least one therapeutic bacterium or spore thereof comprises a strain of lactic acid bacterium or a spore thereof that is isolated from a vegetable source. Also disclosed are methods of production and use of the probiotic compositions.

Description

ELECTRONICALLY TRANSMITTED: SEPTEMBER 10, 2025PROBIOTIC COMPOSITIONS COMPRISING LACTIC ACID BACTERIA AND METHODS FOR PRODUCING AND USING SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The subject application claims benefit under 35 USC § 119(e) of US Provisional Application No. 63 / 692,816, filed September 10, 2024. The entire contents of the above-referenced patent application(s) are hereby expressly incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] Not Applicable.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] The instant application contains, as a separate part of the present disclosure, a Sequence Listing which has been submitted via EFS-Web in computer readable form as an XML file. The Sequence Listing, created September 9, 2025, is named "57910.264wo Sequence Listing. xml" and is 20,572,097 bytes in size. The entire contents of the Sequence Listing are hereby incorporated herein by reference.BACKGROUND

[0004] There is growing interest in improving the overall health and wellbeing of the gut microbiota by using probiotics as a natural and safe approach, especially in relation to aging and stress (Cryan and Dinan, 2012; O'Toole and Jeffery, 2015; Kim et al., 2017; Ticinesi et aL, 2019). The source of probiotic strains is an important factor to consider, as the properties of probiotics can vary depending on the source from which they are isolated (Ray and Didier, 2014; Langkamp-Henken et al., 2015; Kumar et al., 2022; Zhang et al., 2022). Bacteria isolated from organic produce may be a valuable source of probiotics for several reasons. First, organic produce is grown without the use of synthetic fertilizers, pesticides, and other chemicals that may have adverse effects on the microbiota of the produce. This may result in resident microbiota that is more diverse and potentially morebeneficial for human health when consumed. Bacteria isolated from organic produce may have unique properties that make them well-suited for use as probiotics. For example, they may have enhanced survival and colonization abilities in the human gut, or they may produce metabolites that have beneficial effects on gut health (Requena et al., 2018; Kumar et al., 2022; Zhang et al., 2022).

[0005] However, it is important to note that not all bacteria isolated from organic produce may be suitable for use as probiotics. Proper safety and efficacy assessments should be conducted to ensure that any potential probiotic strains are safe for human consumption and have the desired beneficial effects on gut health.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1: Effects of L. lactis and L. mesenteroides combined with CeMbio feeding on the regulation of survival in C. elegans, L. lactis, and L. mesenteroides, resuspended in CeMbio, were tested for the lifespan extension of wild-type N2 worms (p < 0.05, log-rank test). Survival assays were determined in at least three independent experiments (OP50, dark gray line; CeMbio, teal line; CeMbio + L. mesenteroides (Ce + L.m.), blue line; and CeMbio + L. lactis (Ce + L.I.), orange line).

[0007] FIG. 2: Effects of L. lactis and L. mesenteroides and combined with OP50 feeding on the regulation of survival in C. elegans. (A) L. lactis and L. mesenteroides along with resuspended L. lactis and L. mesenteroides in OP50 tested for the lifespan extension of wild-type N2 worms (p < 0.05, logrank test) or the Gehan-Breslow-Wilcoxon test (**, p < 0.05). (B) Resuspended L. lactis and L. mesenteroides in the OP50 lifespan extension of wild-type N2 worms (*, p < 0.05, log-rank test) or the Gehan-Breslow-Wilcoxon test (**, p < 0.05). (C) Monocultures of L. lactis and L. mesenteroides tested for the lifespan extension of wild-type N2 worms (p < 0.05, log-rank test). Survival assays were determined in at least three independent experiments (OP50, dark gray line; OP50 + L. mesenteroides (OP50 + L.m.), blue line; OP50 + L. lactis (OP50 + L.I.), orange line; L. mesenteroides (L.m.), brown line; and L. lactis (L.I.), purple line).

[0008] FIG. 3: Effects of L. lactis and L. mesenteroides and combined with OP50 feeding on the body characteristics of C. elegans. Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner and strain. The color of the bracket indicates the higher significant value of the nested t-test (*, p < 0.05). (A) Length of N2 C. elegans grown on the supplemented bacterial strains. (B) Width of N2 C. elegans grown on the supplemented bacterial strains. (C) Area of the N2 C. elegans grown on the supplemented bacterialstrains. (D) Speed of the N2 C. elegans grown on the supplemented bacterial strains. Body characteristic assays were determined in at least three independent experiments (OP50, dark gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L. lactis (OP50 + L.I.), orange; L. mesenteroides (L.m.), brown; and L. lactis (L.I.), purple).

[0009] FIG. 4: Effects of L. lactis and L. mesenteroides and combined with OP50 feeding on the fecundity of C. elegans. Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner and strain. The color of the bracket indicates the higher significant value of the nested t-test (*, p < 0.05). (A) Average number of total progenies. (B) Average number of progenies per day. Fecundity assays were determined in at least three independent experiments (OP50, dark gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L. lactis (OP50 + L.L), orange; L. mesenteroides (L.m.), brown; and L. lactis (L.I.), purple).

[0010] FIG. 5: Effects of L. lactis and L. mesenteroides and combined with OP50 on the intestinal permeability of wild-type C. elegans. Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner and strain. The color of the bracket indicates the higher significant value of the nested t-test (*, p < 0.05). (A) Control OP50 worms as a visual representation of the Smurf assay. It is detailed with a diagram of the measurement according to the formula. (B) Percentage of the intestinal permeability-based leakage of the blue dye into the body cavity. Smurf assays were determined in at least three independent experiments (OP50, dark gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L. lactis (OP50 + L.L), orange; L. mesenteroides (L.m.), brown; and L. lactis (L.L), purple)

[0011] FIG.6: Effects of L. lactis and L. mesenteroides and combined with OP50 on the reactive oxygen species reporter gcs-lp::GFP. Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner and strain for both the basal and heat-shocked group. The color of the bracket indicates the higher significant value of the nested t-test (*, p < 0.05). Stress assays were determined in at least three independent experiments (OP50, dark gray; OP50 HS, light gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L.m. HS, light blue; OP50 + L. lactis (OP50 + L.L), orange; OP50 + L.L HS, light orange; L. mesenteroides (L.m.), brown; L.m., light brown; L. lactis (L.L), purple; L.L HS, light purple).

[0012] FIG. 7: Effects of L. lactis and L. mesenteroides and combined with OP50 on the cytoplasmic unfolded protein response reporter hsp-16.2p::GFP. Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner and strain for both the basal and heat-shocked group. The color of the bracket indicates the higher significant value of the nested t-test (*, p < 0.05). Stress assays were determined in at least three independent experiments (OP50, dark gray; OP50 HS, light gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L.m. HS, light blue; OP50 + L. lactis (OP50 + L.I.), orange; OP50 + L.l. HS, light orange; L. mesenteroides (L.m.), brown; L.m., light brown; L. lactis (L.L), purple; L.l. HS, light purple).

[0013] FIG. 8: Effects of L. lactis and L. mesenteroides and combined with OP50 on the endoplasmic reticulum unfolded protein response reporter hsp-4::GFP. (A) Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner and strain for both the basal and tunicamycin-treated group. (B) Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner and strain for both the basal and heat-shocked group. The color of the bracket indicates the higher significant value of the nested t-test (*, p < 0.05). Stress assays were determined in at least three independent experiments (OP50, dark gray; OP50 HS, light gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L.m. HS, light blue; OP50 + L. lactis (OP50 + L.L), orange; OP50 + L.l. HS, light orange; L. mesenteroides (L.m.), brown; L.m., light brown; L. lactis (L.L), purple; and L.l. HS, light purple).

[0014] FIG. 9: Effects of L. lactis and L. mesenteroides and combined with OP50 on the endoplasmic reticulum unfolded protein response reporter hsp-6p::GFP. Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner and strain for both the basal and heat-shocked group. The color of the bracket indicates the higher significant value of the nested t-test (*, p < 0.05). Stress assays were determined in at least three independent experiments (OP50, dark gray; OP50 HS, light gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L.m. HS, light blue; OP50 + L. lactis (OP50 + L.L), orange; OP50 + L.l. HS, light orange; L. mesenteroides (L.m.), brown; L.m., light brown; L. lactis (L.L), purple; L.l. HS, light purple).

[0015] FIG. 10: The colonization of L. lactis and L. mesenteroides and combined OP50 in the gut of wild-type C. elegans. This graph shows the CFU from 30 young adults from the different conditionsafter extraction. This shows the colonization of the lactic acid bacteria within the C. elegans gut in 1-day old adults. The colors of the symbols and bars are matched to the treatment (OP50, dark gray circle; OP50 + L. mesenteroides (OP50 + L.m.), blue triangle; OP50 to L. lactis (OP50 + L.I.), orange diamond; L. mesenteroides (L.m.), brown square; and L. lactis (L.I.), purple upside-down triangle).

[0016] FIG. 11: The effects of L. lactis and L. mesenteroides and combined OP50 on the developmental transition of wild-type C. elegans. This graph shows the percent of transition from the fourth larval stage to young adult in hours showing no significant difference in development time. Experiment comprised of four independent samples with a cumulative n > 300 for each condition. The color of the bar is matched to the treatment (OP50, dark gray; OP50 + L. mesenteroides (OP50 + L.m.) blue; OP50 to L. lactis (OP50 + L.l.) orange; L. mesenteroides (L.m.) brown; and L. lactis (L.l.) purple).

[0017] FIG. 12: Representative images for the effects of L. lactis and L. mesenteroides and combined OP50 on the body morphology of wild-type C. elegans. The color of the bracket is matched to FIG. 3. Left to right: first image, OP50 (dark gray); second image, OP50 + L. mesenteroides (OP50 + L.m.), blue; third image, OP50 to L. lactis (OP50 + L.L), orange; fourth image, L. mesenteroides (L.m.), brown; and fifth image, L. lactis (L.L), purple.

[0018] FIG. 13: Representative images for the effects of L. lactis and L. mesenteroides and combined OP50 on the reactive oxygen species reporter gcs-lp::G FP. Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner, and strain for both the basal and heat shocked group. The color of the bracket is matched to FIG. 6. OP50, dark gray; OP50 HS, light gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L.m. HS, light blue; OP50 to L. lactis (OP50 + L.L), orange; OP50 + L.l. HS, light orange; L. mesenteroides (L.m.), brown; L.m. HS, light brown; L. lactis (L.L), purple; L.l. HS, light purple; and positive control, gold.

[0019] FIG. 14: Representative images for the effects of L. lactis and L. mesenteroides and combined OP50 on the reactive oxygen species reporter hsp-16.2p::GFP. Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner, and strain for both the basal and heat shocked group. The color of the bracket is matched to FIG. 7. OP50, dark gray; OP50 HS, light gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L.m. HS, light blue; OP50 to L. lactis (OP50 + L.L), orange; OP50 + L.l. HS, light orange; L.mesenteroides (L.m.), brown; L.m. HS, light brown; L. lactis (L.I.), purple; L.l. HS, light purple; and positive control, gold.

[0020] FIG. 15: Representative images for the effects of L. lactis and L. mesenteroides and combined OP50 on the reactive oxygen species reporter hsp-4.•:GFP. Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner, and strain for both the basal. A.) tunicamycin, or B.) heat shocked. The color of the bracket is matched to FIG. 8. OP50, dark gray; OP50 HS, light gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L.m. HS, light blue; OP50 to L. lactis (OP50 + L.L), orange; OP50 + L.l. HS, light orange; L. mesenteroides (L.m.), brown; L.m. HS, light brown; L. lactis (L.L), purple; L.l. HS, light purple; and positive control, gold.

[0021] FIG. 16: Representative images for the effects of L. lactis and L. mesenteroides and combined OP50 on the reactive oxygen species reporter hsp-6p::GFP. Cumulative groups of the five combinations were analyzed in relation to the control, OP50, and its related supplementation partner, and strain for both the basal and heat shocked group. The color of the bracket is matched to FIG. 9. OP50, dark gray; OP50 HS, light gray; OP50 + L. mesenteroides (OP50 + L.m.), blue; OP50 + L.m. HS, light blue; OP50 to L. lactis (OP50 + L.L), orange; OP50 + L.l. HS, light orange; L mesenteroides (L.m.), brown; L.m. HS, light brown; L. lactis (L.L), purple; L.L HS, light purple; and positive control, gold.

[0022] FIG. 17: acid tolerance assay of LAB (L. mesenteroides) growth in MRS adjusted to pH 3.0 with HCI.

[0023] FIG. 18: bile tolerance assay of LAB L. mesenteroides to bile at 0.3%.

[0024] FIG. 19: hemolysis assay of LAB L. mesenteroides and P. aeruginosa PA14.DETAILED DESCRIPTION

[0025] Before explaining at least one embodiment of the inventive concept(s) in detail by way of exemplary language and results, it is to be understood that the inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components set forth in the following description. The inventive concept(s) is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary - notexhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0026] Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0027] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this presently disclosed inventive concept(s) pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0028] All of the compositions and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the inventive concept(s) have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the inventive concept(s). All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the inventive concept(s) as defined by the appended claims.

[0029] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0030] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the"include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0031] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0032] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0033] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0034] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or threepercent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0035] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0036] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0037] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. For example, the term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.

[0038] As used herein, the phrases "associated with" and "coupled to" include both direct association / binding of two moieties to one another as well as indirect association / binding of two moieties to one another.

[0039] The term "pharmaceutically acceptable" refers to compounds and compositions which are suitable for administration to humans and / or animals without undue adverse side effects, such as (but not limited to) toxicity, irritation, and / or allergic response, commensurate with a reasonable benefit / risk ratio.

[0040] The term "patient" or "subject" as used herein includes human and veterinary subjects. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including (but not limited to) humans, domestic and farm animals, nonhuman primates, and any other animal that has mammary tissue.

[0041] The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include, but are not limited to, individuals already having a particular condition / disease / infection as well as individuals who are at risk of acquiring a particular condition / disease / infection (e.g., those needing prophylactic / preventative measures). The term "treating" refers to administering an agent to a subject / patient for therapeutic and / or prophylactic / preventative purposes.

[0042] A "therapeutic composition" or "pharmaceutical composition" refers to an agent that may be administered in vivo to bring about a therapeutic and / or prophylactic / preventative effect.

[0043] The terms "administration" and "administering," as used herein, will be understood to include all routes of administration known in the art. In addition, the compositions of the present disclosure (and / or the methods of administration of same) may be designed to provide delayed, controlled, or sustained release using formulation techniques which are well known in the art.

[0044] The term "pharmaceutically acceptable carrier or excipient" includes any carriers or excipients known in the art may be utilized in accordance with the present disclosure. For example (but not by way of limitation), a physiological compatible carrier (e.g., saline) that is compatible with maintaining the structure / activity of the active ingredient(s) when administered, and compatible with the desired mode of administration, may be utilized as the pharmaceutically acceptable carrier in accordance with the present disclosure. In addition, the active ingredient(s) may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient(s). Suitable excipients include, for example but not by way of limitation, water, saline, dextrose, glycerol, ethanol, and the like, or any combination thereof.

[0045] As used herein, the terms "food," "food product," and "food composition" refer to a product or composition that is intended for oral ingestion by a human or other mammal and comprises at least one nutrient for the human or other mammal.

[0046] "Nutritional compositions" and "nutritional products," as used herein, include any number of food ingredients and possibly optional additional ingredients based on a functional need in the product and in full compliance with all applicable regulations. The optional ingredients mayinclude, but are not limited to, conventional food additives, such as (but not limited to) acidulants, additional thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifies, excipient, flavor agent, mineral, osmotic agents, a pharmaceutically acceptable carrier, preservatives, stabilizers, sugar, sweeteners, texturizers, vitamins, and the like, as well as any combinations thereof. The optional ingredients can be added in any suitable amount.

[0047] The term "probiotic" refers to live, non-pathogenic microorganisms, e.g., bacteria, microbial cell preparations, or components of microbial cells which can confer health benefits to a host organism. (Salminen S, Ouwehand A. Benno Y. et al "Probiotics: how should they be defined" Trends Food Sci. Technol. 1999:10 107-10). Some species, strains, and / or subtypes of non- pathogenic bacteria are currently recognized as probiotic. The probiotic may be a variant or a mutant strain of bacterium. Probiotic bacteria may be naturally mutated or genetically engineered modified to retain, enhance or improve desired biological properties, e.g., survivability to provide probiotic properties or to retain, enhance or improve probiotic properties.

[0048] The terms "derived from," "derivative," "variant," "mutant" (e.g., "mutant strain"), or any grammatical variant thereof, as used herein, refer to a component that is isolated from or made using a specified molecule / substance (e.g., a strain of the present disclosure). For example, a bacterial strain that is derived from a first bacterial strain (e.g., a deposited strain) can be a strain that is identical or substantially similar to the first strain. In the case of bacterial strains, the derived strain can be obtained by, e.g., naturally occurring mutagenesis, artificially directed mutagenesis, artificially random mutagenesis or othergenetic engineering techniques, and it retains, enhances or improves at least one ability of the deposited strain.

[0049] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition disclosed herein in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage form depend on the particular compounds employed, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0050] Turning now to the inventive concepts, the present disclosure is related to therapeutic compositions, systems, kits, and methods of producing and using same. In particular, the present disclosure is related to compositions, systems, kits, and methods that contain one or more lactic acid bacteria (LAB) derived from a vegetable source, such as, but not limited to, Lactococcus lactisand Leuconostoc mesenteroides. These LABs derived from a vegetable source differ structurally from their counterparts that are derived from dairy sources, with in some cases, at least about 10% divergence between the genomic sequences of the two strains. The compositions of the present disclosure are demonstrated herein to be particularly effective for use in subjects with an altered gut microbiome, including subjects undergoing (or having recently completed) antibiotic treatment; patients experiencing at least one gastrointestinal issue; cancer patients; immunocompromised patients; patients undergoing immunotherapy, chemotherapy, and / or radiation therapy; and the like; or any other condition or disorder that results in an unhealthy gut biome. Further, the compositions of the present disclosure are demonstrated herein to be advantageously effective as a food additive; in particular (but not by way of limitation), the compositions have surprisingly and unexpectedly been shown to extend a life span of a model organism (C. elegans) in the examples presented herein.

[0051] Certain non-limiting embodiments of the present disclosure are directed to a probiotic composition and / or food additive that comprises an effective amount of at least one therapeutic bacterium or a spore thereof. In specific (but non-limiting) embodiments, the at least one therapeutic bacterium or spore thereof comprises a strain of lactic acid bacterium or a spore thereof that is isolated from a vegetable source. Any vegetable sources known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure. In particular (but non-limiting) embodiments, the vegetable source is an organic herb, such as (but not limited to) basil.

[0052] The compositions of the present disclosure may be formulated for administration to a subject (such as, but not limited to, a human and / or an animal) via any formulation known in the art or otherwise contemplated herein that may be acceptable to the subject for consumption. For example (but not by way of limitation), the compositions may be a nutritional composition (such as, but not limited to, a food product, an animal feed, or a medical food) or a supplement (which can be administered by itself or added to food and / or water).

[0053] The probiotic compositions of the present disclosure may include at least one lactic acid bacterium / spore, or more than one lactic acid bacteria / spore thereof obtained from a vegetable source, such as (but not limited to), at least two lactic acid bacteria / spores thereof, at least three lactic acid bacteria / spores thereof, at least four lactic acid bacteria / spores thereof, at least five lactic acid bacteria / spores thereof, and the like.

[0054] When at least two strains of lactic acid bacteria are present in the probiotic composition, the two strains may be present at any weight ratio that allows the probiotic composition to function in accordance with the present disclosure. For example, but not by way of limitation, the two strains may be present at a weight ratio of about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1.5:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, and the like, as well as a range of weight ratios formed from two of any of the above values, such as (but not limited to), about 20:1 to about 1:20, about 10:1 to about 1:1, about 1:1 to about 1:10, about 10:1 to about 2:1, about 8:1 to about 2:1, about 7:1 to about 2:1, about 6:1 to about 2:1, about 5:1 to about 3:1, and the like.

[0055] Particular (but non-limiting) examples of lactic acid bacteria isolated from vegetable sources that may be utilized in accordance with the present disclosure include Lactococcus lactis (L. lactis) and Leuconostoc mesenteroides (L. mesenteroides). In a non-limiting embodiment, each strain is isolated from basil. For example (but not by way of limitation), a L. mesenteroides strain isolated from basil has a genomic sequence comprising at least a portion of at least one of SEQ ID NOS:l-6, and a L. lactis isolated from basil has a genomic sequence comprising at least a portion of at least one of SEQ ID NOS:7-12. Alternatively, the L. mesenteroides strain isolated from basil has a genomic sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to at least a portion of at least one of SEQ ID NOS:l-6, and / or the L. lactis isolated from basil has a genomic sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to at least one of SEQ ID NOS:7-12.

[0056] In a particular (but non-limiting) embodiment, the strain of lactic acid bacterium or a spore thereof of the probiotic compositions comprises at least one a-hemolysin.

[0057] In a particular (but non-limiting) embodiment, the probiotic composition comprises both L. lactis and L. mesenteroides. In particular, but non-limiting, embodiments, the probiotic composition comprises L. mesenteroides and L. lactis present at any of the weight ratios discussedin detail herein above. For example (but not by way of limitation), L. mesenteroides and L. lactis may be present at a weight ratio in a range of from about 20:1 to about 1:20, a weight ratio in a range of from about 10:1 to about 1:10, a weight ratio in a range of from about 10:1 to about 1:1, a weight ratio in a range of from about 6:1 to about 2:1, a weight ratio in a range of from about 5:1 to about 3:1, a weight ratio of about 4:1, and the like.

[0058] The probiotic compositions of the present disclosure may contain one or more additional ingredients as discussed or otherwise contemplated herein. Non-limiting examples of additional ingredients include pharmaceutically acceptable carriers or excipients, prebiotics, formulary ingredients, antibiotics and other concomitantly administered active / therapeutic ingredients, and the like, as well as any combinations thereof.

[0059] In certain particular (but non-limiting) embodiments, the probiotic compositions may further include at least one pharmaceutically acceptable carrier or excipient. Non-limiting examples of pharmaceutically acceptable carriers or excipients that may be utilized in accordance with the present disclosure include hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose acetate succinate, microcrystalline cellulose, milk powder, trehalose, xanthan gum, banana powder, dextran, maltose, lactose, glutamic acid, glycerin, PBS, mannitol, partially pregelatinized starch, silicon dioxide, magnesium stearate, maltodextrin, functionalized mineral (Omya), PEARLITOL® DC (Direct compression grade mannitol, , Roquette Freres Corporation, Lestrem, Cedex, France), polyvinylpyrrolidone (PVP), dibasic calcium phosphate (DCP), a pharmaceutically acceptable inhalant solvent, and the like, as well as any combinations thereof.

[0060] In certain particular (but non-limiting) embodiments, the probiotic compositions may further include at least one prebiotic. Any type of prebiotic known in the art or otherwise contemplated herein may be utilized, including (but not limited to) various types of fibers (carbohydrates), various types of sugars for fermentation, various types of acid for pH regulation, various short-chain fatty acids, and the like. Non-limiting examples of specific prebiotics that may be utilized in accordance with the present disclosure include |3-glucan, galacto-oligosaccharides, fructo-oligosaccharides, starch, trans-galacto-oligosaccharides, gluco-oligosaccharides, glyco- oligosccharides, lactulose, lactitol, malto-oligosaccharides, xylo-oligosaccharides, stachyose, raffinose, inulin, isomalto-oligosaccharides, xylo-oligosaccharides, lacticol, lactulose, cereal fibre, short-chain oligosaccharide, and combinations thereof.

[0061] In addition, the strain(s) of lactic acid bacteria or spore(s) thereof may be treated by any methods known in the art during formulation of the probiotic compositions. Certain types of treatments may be utilized to increase the lifespan of the bacteria and / or ensure delivery of the bacteria through the Gl tract and to the appropriate portion of the intestine. For example (but not by way of limitation), the bacteria / spores may be lyophilized, encapsulated, and / or heat treated, and the like, as well as any combination thereof. Further (and / or in addition thereto), the bacteria / spores may be treated so as to increase the acid tolerance and / or bile tolerance of the bacteria / spores.

[0062] When the bacteria / spores are encapsulated, any encapsulation method known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure. Nonlimiting examples thereof include microencapsulation (such as for use in chocolate, bakery items, etc.), polymeric encapsulation, spray drying, emulsion, extrusion, spray freeze drying, layer by layer, ionic gelation, vibration technology, sol-gel encapsulation, cyclodextrin inclusion, solvent evaporation method, and the like, as well as any combinations thereof.

[0063] Various methods for biopolymer encapsulation for delivery of probiotics are known in the art and described, for example (but not by way of limitation), in Vijayaram et al. (AIMS Microbiology (2024) 10(4):986-1023, incorporated herein by reference).

[0064] The probiotic compositions of the present disclosure may be provided in any form and formulation for administration in any manner that allows the probiotic compositions to function in accordance with the present disclosure. For example (but not by way of limitation), the compositions may be in solid form (such as, but not limited to, capsules, chewables, powders, sachets, and dry powder inhalers or nasal products), liquid form (such as, but not limited to, emulsions, microemulsions, solutions, suspensions, syrups, and elixirs), or gels (such as, but not limited to, gummies), sprays, mists, and / or aerosols. Alternatively, the compositions may be in the form of a baked good(s) or other type of food product(s); non-limiting examples thereof include yogurt, kimchi, dairy products, fermented foods, yakult drink, cheese, chocolate, bakery items, and the like, as well as combinations thereof.

[0065] The probiotic compositions may be administered alone or in combination with one or more additional therapies and may be administered by a variety of administration routes. The particular mode selected will depend, of course, upon the probiotic(s) selected, the condition being treated, the severity of the condition, whether the treatment is therapeutic or prophylactic, and thedosage required for efficacy. The methods of the present disclosure, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active probiotics without causing clinically unacceptable adverse effects. Non-limiting examples of administration routes that may be utilized in accordance with the present disclosure include oral, transdermal, parenteral, subcutaneous, intranasal, intratracheal, intrabronchial, mucosal, intramuscular, intraperitoneal, intravitreal, and / or intravenous routes, and the like.

[0066] In certain particular (but non-limiting) embodiments, the probiotic compositions are formulated for oral and / or inhalant administration.

[0067] In certain non-limiting embodiments, the at least one therapeutic bacterium or spore thereof of the probiotic composition may be at least partially resistant to at least one antibiotic. In certain particular (but non-limiting) embodiments, the at least one therapeutic bacterium or spore thereof of the probiotic composition is substantially resistant to at least one antibiotic.

[0068] Certain non-limiting embodiments of the present disclosure are directed to methods that comprise administering one or more of any of the probiotic compositions disclosed or otherwise contemplated herein to a subject in need of treatment. The subject may be (for example, but not by way of limitation) a mammal, such as a human or a domestic animal.

[0069] The subject may have any condition for which treatment with one of the probiotic compositions of the present disclosure would be desired. The subject may be experiencing at least one gastrointestinal (Gl) symptom, such as (but not limited to) an upset stomach, heartburn, nausea, vomiting, loose bowel movements, and the like, as well as combinations thereof.

[0070] Alternatively (and / or in addition thereto), the subject may have various conditions, diseases and / or disorders, and / or undergone various treatments that are known or may be suspected of affecting the gut biome. Non-limiting examples thereof include treatment with an antibiotic, chemotherapeutic agent, and / or immunotherapeutic agent; radiation treatment; a gastrointestinal tract disease or condition; cancer; an autoimmune disease; as well as any disease, disorder, or condition that results in the patient being immunocompromised.

[0071] The probiotic compositions of the present disclosure may be administered in therapeutically effective amounts. An effective amount is a dosage of the probiotic composition sufficient to provide a therapeutically or medically desirable result or effect in the subject to which the composition is administered. The effective amount will vary with the particular condition beingtreated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent or combination therapy (if any), the specific route of administration, and like factors within the knowledge and expertise of the health practitioner.

[0072] Generally, a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the nature and extent of condition / disease in the subject, all of which can be determined by one of ordinary skill in the art. The dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. Non-limiting examples of therapeutically effective amounts include amounts in a range from about 0.1 mg to about 1 g per daily total dose, or a range of from about 1 mg to about 500 mg per daily total dose, or a range of from about 5 mg to about 200 mg per daily total dose, or a range of from about 1 mg to about 100 mg per daily total dose, or a range of from about 5 mg to about 100 mg per daily total dose, or a range of from about 1 mg to about 75 mg per total daily dose, or a range of from about 5 mg to about 75 mg per daily total dose, or a range of from about 1 mg to about 60 mg per daily total dose, or a range of from about 5 mg to about 60 mg per daily total dose, or a range of from about 1 mg to about 50 mg per daily total dose, or a range of from about 5 mg to about 50 mg per daily total dose, or the like.

[0073] In addition, the probiotic compositions of the present disclosure may be present at any therapeutically effective amounts of CPU per daily dosage known in the art or otherwise contemplated herein. Non-limiting daily dosages include about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 100 million, about 200 million, about 300 million, about 400 million, about 500 million, about 600 million, about 700 million, about 800 million, about 900 million, about 1 billion, about 5 billion, about 10 billion, about 20 billion, about 30 billion, about 40 billion, about 50 billion, about 60 billion, about 70 billion, about 80 billion, about 90 billion, about 100 billion, about 110 billion, about 120 billion, about 130 billion, about 140 billion, about 150 billion, about 160 billion, about 170 billion, about 180 billion, about 190 billion, about 200 billion, about 210 billion, about 220 billion, about 230 billion, about 240 billion, about 250 billion, about 260 billion, about 270 billion, about 280 billion, about 290 billion, about 300 billion, about 310 billion, about 320 billion, about 330 billion, about 340 billion, about 350 billion, about 360 billion, about 370 billion, about 380 billion, about 390 billion, about 400 billion, about 410 billion, about 420 billion, about 430 billion, about 440 billion, about 450 billion, about 460 billion,about 470 billion, about 480 billion, about 490 billion, about 500 billion, about 510 billion, about 520 billion, about 530 billion, about 540 billion, about 550 billion, about 560 billion, about 570 billion, about 580 billion, about 590 billion, about 600 billion, about 610 billion, about 620 billion, about 630 billion, about 640 billion, about 650 billion, about 660 billion, about 670 billion, about 680 billion, about 690 billion, about 700 billion, about 710 billion, about 720 billion, about 730 billion, about 740 billion, about 750 billion, about 760 billion, about 770 billion, about 780 billion, about 790 billion, about 800 billion, and the like, as well as a range formed from two of any of the above values (i.e., a range of from about 100 million to about 800 billion, a range of from about 500 million to about 800 billion, a range of from about 1 billion to about 700 billion, a range of from about 100 billion to about 700 billion, a range of from about 200 billion to about 600 billion, and the like).

[0074] If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six, or more sub-doses, for example, administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the probiotic compositions are administered for more than 7 days, more than 10 days, more than 14 days, or more than 21 days. In still other embodiments, the probiotic composition is administered over a period of weeks or months. In still other embodiments, the probiotic composition is delivered on alternate days. For example, the probiotic composition may be delivered every two days, or every three days, or every four days, or every five days, or every six days, or every week, or every month.

[0075] The methods of the present disclosure may optionally include one or more additional steps, such as administration of one or more additional active agents either simultaneously or wholly or partially sequentially with the probiotic compositions of the present disclosure. For example (but not by way of limitation), the method may further include the step of administering at least one antibiotic to the subject, wherein the at least one therapeutic bacterium or spore thereof of the probiotic composition is at least partially or substantially resistant to the at least one antibiotic. In this manner, the probiotic compositions can help restore a healthy gut microbiome that is altered by the antibiotic administration.

[0076] Certain non-limiting embodiments of the present disclosure are directed to food additives that comprise at least one of any of the probiotic compositions or strains of lactic acid bacteria (or spores thereof) isolated from a vegetable source, as disclosed or otherwise contemplated herein. In particular (but non-limiting) embodiments, the food additive may beutilized as a food additive for a model organism, such as (but not limited to) C. elegans, rats, mice, rabbits, pigs, hamsters, guinea pigs, dogs, primates, and the like.

[0077] Certain non-limiting embodiments of the present disclosure are directed to methods that comprise the step of administering one or more of any of the food additives disclosed herein to a model organism or subject.

[0078] As disclosed in the Examples below, it has been shown herein that the food additives of the present disclosure exhibit a surprising and unexpected advantage; addition of one or more of the strains of lactic acid bacteria isolated from a vegetable source as disclosed herein to a normal diet for the model organism has been shown to extend the life span of the model organism when compared to a life span of a model organism fed the normal diet in the absence of the food additive. Therefore, the compositions of the present disclosure affect not only the gut microbiome of the subject to which they are administered, but can also extend the life span of the subject as well.EXAMPLES

[0079] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein after. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.Example 1

[0080] The short lifespan of Caenorhabditis elegans enables the efficient investigation of probiotic interventions affecting stress and longevity involving the potential therapeutic value of Lactococcus lactis and Leuconostoc mesenteroides isolated from organic basil. The lactic acid bacteria were cultured from the produce collected from a local grocery store in Tulsa, Oklahoma, and then identified through 16S rDNA sequencing and biochemical tests. To dive deep into this analysis for potential probiotic therapy, fluorescent reporters were used that allow for assessment of the differential induction of multiple stress pathways such as oxidative stress and the cytoplasmic, endoplasmic reticulum, and the mitochondrial unfolded protein response. This is combined with the classic health span measurements of survival, development, and fecundity, allowing a wide range of organismal observations of the different communities of microbes supported by probiotic supplementation with Lactococcus lactis and Leuconostoc mesenteroides. These strains wereinitially assessed in relation to the Escherichia coli feeding strain OP50 and the C. elegans microbiome. The supplementation showed a reduction in the median lifespan of the worms colonized within the microbiome. This was unsurprising, as negative results are common when probiotics are introduced into healthy microbiomes. To further assess the supplementation potential of these strains on an unhealthy (undifferentiated) microbiome, the typical axenic C. elegans diet, OP50, was used to simulate this single-species biome. The addition of lactic acid bacteria to OP50 led to a significant improvement in the median and overall survival in simulated biomes, indicating their potential in probiotic therapy. The study analyzed the supplemented cultures in terms of C. elegans' morphology, locomotor behavior, reproduction, and stress responses, revealing unique characteristics and stress response patterns for each group. As the microbiome's influence on the health span gains interest, the present disclosure aimsto understand the microbiome relationships that result in differential stress resistance and lifespans by supplementing microbiomes with Lactococcus lactis and Leuconostoc mesenteroides isolated from organic basil in C. elegans.

[0081] 1 Introduction

[0082] There is growing interest in improving the overall health and wellbeing of the gut microbiota by using probiotics as a natural and safe approach, especially in relation to aging and stress (Cryan and Dinan, 2012; O'Toole and Jeffery, 2015; Kim et al., 2017; Ticinesi et aL, 2019). The source of probiotic strains is an important factor to consider, as the properties of probiotics can vary depending on the source from which they are isolated (Ray and Didier, 2014; Langkamp-Henken et al., 2015; Kumar et al., 2022; Zhang et al., 2022). Bacteria isolated from organic produce may be a valuable source of probiotics for several reasons. First, organic produce is grown without the use of synthetic fertilizers, pesticides, and other chemicals that may have adverse effects on the microbiota of the produce. This may result in resident microbiota that is more diverse and potentially more beneficial for human health when consumed. Bacteria isolated from organic produce may have unique properties that make them well-suited for use as probiotics. For example, they may have enhanced survival and colonization abilities in the human gut, or they may produce metabolites that have beneficial effects on gut health (Requena et al., 2018; Kumar et al., 2022; Zhang et al., 2022).

[0083] However, it is important to note that not all bacteria isolated from organic produce may be suitable for use as probiotics. Proper safety and efficacy assessments should be conducted to ensure that any potential probiotic strains are safe for human consumption and have the desiredbeneficial effects on gut health. In this study, we investigate the probiotic potential of two strains isolated from Oklahoma-grown organic basil, positively identified through 16S rRNA gene sequencing, Lactococcus lactis (L. loctis) and Leuconostoc mesenteroides (L. mesenteroides) (Van Tieghem, 1878; Schleifer et al., 1985).

[0084] A well-established model organism that has been used extensively in aging and stress research is Caenorhabditis elegans (C. elegans) (Lithgow et al., 1995; Gems and Riddle, 2000; Lithgow and Walker, 2002; Panowski and Dillin, 2009; Kenyon, 2010; Gems and Partridge, 2013). One advantage of using C. elegans as a model for evaluating probiotic therapy is its short lifespan, which allows for the rapid screening of potential therapeutic interventions (Brenner, 1974; Kumar et al., 2022; Zhang et al., 2022). In addition, C. elegans has a well-characterized genome and is genetically tractable (C. elegans Sequencing Consortium, 1998; Howe et al., 2016), making it a useful tool for studying the mechanisms underlying the effects of probiotics on host stress responses and aging (Riera et al., 2014; O'Toole and Jeffery, 2015).

[0085] Studies in C. elegans have demonstrated that probiotics can improve stress responses and increase the lifespan (Grompone et al., 2012; Martorell et al., 2016; Kumar et al., 2022; Zhang et al., 2022). For example, the administration of Lactobacillus rhamnosus CNCM 1-3690 has been shown to improve survival in C. elegans exposed to oxidative stress (Grompone et al., 2012), and the administration of Lactobacillus plantarum JBC5 and Lactobacillus fermentum strain JDFM216 has been shown to extend the lifespan in C. elegans (Park et al., 2018; Kumar et al., 2022).

[0086] Importantly, the gut microbiota of C. elegans has been shown to play a crucial role in mediating the effects of probiotics on stress responses and aging (Cabreiro and Gems, 2013; Oh et al., 2015; Martorell et al., 2016; Kissoyan et al., 2019; Yang et aL, 2019; Dirksen et al., 2020; Poupet et al., 2020; Kumar et al., 2022; Yun et al., 2022). For example, the administration of the probiotic Bifidobacterium animalis subsp. lactis CECT 8145 reduced fat and mobilized lipids for the metabolism, while modulating the antioxidant response in C. elegans (Martorell et al., 2016). However, this effect was dependent on the composition of the differentiated gut microbiota present in the C. elegans intestine (Martorell et aL, 2016).

[0087] The stress responses modulated by probiotic supplementation are a measure of suitability when assessing beneficial probiotic therapy. A few of those measures include the cytoplasmic (cyt), endoplasmic reticulum (ER), and mitochondrial (mt) unfolded protein responses (UPRs), which are important parameters that can be used to evaluate the potential effects ofprobiotic supplementation in C. elegans (Yoneda et aL, 2004; Gardner and Walter, 2011; Hetz and Papa, 2018; Kim and Kim, 2018; Martucciello et aL, 2020). The UPRcyt, UPRER, and UPRmt are cellular stress responses, which are activated during a disruption in protein folding and quality control in the cytoplasm, ER, or mitochondria that can contribute to the stress response in the course of aging and disease (Morley et aL, 2002; Cohen and Dillin, 2008; Hipp et aL, 2014; Balchin et aL, 2016; Maulik et aL, 2017; Mamun et al., 2020). However, if probiotic supplementation is found to positively modulate UPRcyt, UPRER, and UPRmt activity, it indicates that probiotics can promote improved cytoplasmic, ER, or mitochondrial health or the ability to handle a stress event (Kim and Kim, 2018; Kumar et al., 2022). This, in turn, could have implications for human health, as cytoplasm, ER, or mitochondrial protein quality control is important for maintaining cellular function and preventing the accumulation of misfolded proteins that can lead to diseases such as Parkinson's disease, cystic fibrosis, or Alzheimer's disease (Morley et aL, 2002; Cohen and Dillin, 2008; Hipp et aL, 2014; Balchin et aL, 2016; Mamun et aL, 2020).

[0088] Studies have also shown that probiotics can modulate UPRcyt, UPRER, and UPRmt in C. elegans. One example of probiotic regulation in C. elegans uses the UPRcyt marker, hsp-16.2, after supplementation with Lacticaseibacillus rhamnosus Probio-M9, which observed no increase in hsp- 16.2 expression (Zhang et aL, 2022). This indicates that probiotic supplementation modulates the observed lifespan extension through an hsp-16.2 independent mechanism. The UPRER was also not modulated by Lacticaseibacillus rhamnosus Probio-M9, shown by the normal expression of hsp-4, a C. elegans marker of ER UPR stress (Zhang et aL, 2022; Zhang et aL, 2022). However, Lacticaseibacillus rhamnosus Probio-M9 modulates hsp-6, a C. elegans marker of UPRmt, consistent with the induction of UPRmt stress (Zhang et aL, 2022). Studies extending outside of Lacticaseibacillus rhamnosus Probio-M9 into potential probiotic strains such as Lacticaseibacillus rhamnosus strain GG, Lactobacillus rhamnosus CNCM 1-3690, or Lactobacillus plantarum JBC5 come to alternative opinions on whether the mechanistic benefits are positive or negative for lifespan extension, promotion of mitochondrial health, and stress response with probiotic colonization with the induction of a stress response (Grompone et aL, 2012; Kumar et al. , 2022; Yun et aL, 2022; Zhang et al., 2022). The gain or loss of a stress response cannot be assessed in isolation. The stress event needs to be considered along with other endpoint factors such as lifespan extension to assess a positive or negative mechanistic outcome associated with probiotic supplementation.

[0089] As such, other important parameters will be used to gage the potential effects of probiotic supplementation in C. elegans such as survival, fecundity, and development. C. elegans' short lifespan enables efficient study of interventions affecting survival (Brenner, 1974; Grompone et al., 2012; Martorell et al., 2016; Zhou et al., 2021; Zhou et al., 2022; Kumar et al., 2022; Liu et al., 2022; Yun et al., 2022; Zhang et al., 2022). If probiotic supplementation is found to increase the survival of C. elegans, it indicates that probiotics promote better health and longevity. There are many studies that indicate that probiotic supplementation can have beneficial effects on the survival of C. elegans under various stress conditions (heat, oxidative, etc.) and may increase the lifespan under normal conditions as well (Grompone et al., 2012; Oh et al., 2015; Martorell et al., 2016; Zhou et aL, 2021; Zhou et al., 2022; Kumar et al., 2022; Yun et aL, 2022; Zhang et al., 2022). Changes in fecundity can be indicative of alterations in the host's overall health, such as oxidative stress, inflammation, or altered metabolism (Kumar et al., 2022; Zhang et al., 2022). However, it is important to mention that the effects of probiotics on survival, fecundity, and development may depend on the specific probiotic strains used, the timing and duration of supplementation, and other experimental factors, such as the ability to survive and colonize the gut (Grompone et al., 2012; Kumar et al., 2022; Yun et al., 2022; Zhang et al., 2022).

[0090] We investigate the probiotic potential of L. lactis and L. mesenteroides supplementation on an unhealthy microbiome, simulated by the typical axenic C. elegans diet, E. coli (OP50) (Brenner, 1974; Cabreiro and Gems, 2013). Our efforts to assess oxidative, UPRcyt, UPRER, and UPRmt stress were achieved by observing gcs-1, hsp-16.2, hsp-4, and hsp-6 stress reporters, respectively, combined with classic health span measurements of survival, fecundity, and development for a wide range of organismal observations of the newly differentiated microbiome (Cabreiro and Gems, 2013; Detienne et al., 2016; Manjarrez and Mailler, 2020; Zhou et aL, 2021; Annapure and Nair, 2022; Kumar et al., 2022; Yun et al., 2022; Zhang et aL, 2022). The influence of the microbiome on health span is a growing area of interest, with the recent chemotherapeutic advances with fecal transplants efficiently conferring sensitivity to known treatments (Davar et aL, 2021). C. elegans lacks many of the complex physiological systems found in humans; however, this does not limit the potential of C. elegans as a model for probiotic therapy, as it provides valuable insights into many fundamental mechanisms underlying the beneficial effects of probiotics on various metabolic and neurodegenerative diseases (Sonnenburg and Backhed, 2016; Kim et aL, 2017; Grumezescu andHolban, 2018; Mangiola et aL, 2018; Requena et al., 2018; Ticinesi et al., 2019; Annapure and Nair, 2022; Czyz, 2022; Ling et al., 2022; Wang and Zheng, 2022; Marotta, 2023).

[0091] 2 Materials and methods

[0092] 2.1 Culturing Caenorhabditis elegans

[0093] The C. elegans strains used in this study are listed in Table 1. Worms were cultured at 20°C on a nematode growth medium (NGM) agar (Brenner, 1974). Plates were seeded with precultured bacterial strains according to the probiotic supplementation method. C. elegans were age- synchronized using the egg laying technique and incubated at 20°C until the larvae reached the desired stage of development for subsequent experimentation.

[0094] 2.2 Probiotic supplementation

[0095] The bacterial strains used in this study are listed in Table 2. The lactic acid bacteria Lactococcus lactis and Leuconostoc mesenteroides were isolated from organic basil using standard isolation protocols for lactic acid bacteria.

[0096] A solution of the probiotic supplement Lactococcus lactis and Leuconostoc mesenteroides was prepared in liquid NGM buffer. The culture was grown overnight at 35°C, concentrated, and resuspended at 15.24 mg / mL. The probiotic solution was added to OP50 at 10% (w / v) and seeded on NGM agar plates using a final concentration of 8 mg / mL. CeMbio cultures were prepared according to the previously designed methods (Dirksen et al., 2020) and seeded on NGM agar plates according to the protocol mentioned previously.

[0097] 2.3 Survival analysis

[0098] All survival analysis were performed at 20°C. The L4 stage worms were transferred to fresh plates and used on day 3 for the survival assay (Amrit et al., 2014). The worms were transferred every day until they ceased producing progeny, after approximately 3-5 days and then every 2 days until all worms died, unless indicated otherwise (the plates were spotted for use every 2 days from fresh cultures). For each experiment, at least three plates (25 worms per plate) per bacterial strain were analyzed for the CeMbio survival analysis, and for OP50 supplementation experimentation, five plates (at least 25 worms per plate) per bacterial strain were analyzed. A death event was determined via ceased pharyngeal pumping and no response to gentle prodding with a platinum worm pick. The worms were examined daily. If the worms were unintentionally lost, AVID (age- associated vulval integrity defects frequently described as ruptured) (Leiser et aL, 2016), or had undergone matricide, these were censored and excluded from the survival analysis. Statisticalanalyses were performed using GraphPad Prism 9.5.1 for statistical log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon analysis, in all cases p < 0.05 was considered significant.

[0099] 2.4 Fecundity, body characteristics, and locomotion

[0100] Fecundity was measured with five individual L4 synchronized hermaphrodites (five repeats / 25 worms in total / bacterial composition). Each individual adult was transferred to fresh plates daily (one worm per plate) until reproduction ceased. The total number of viable offspring was counted per day per worm.

[0101] Body characteristics and locomotion were measured from three plates of (at least 20) age-synchronized worms per bacterial strain, at day 1 of adulthood. Videos were taken using a stereo microscope (Nikon S74747) with a D1000 camera and then analyzed using Worm Lab software (MBF Bioscience). The software analyzed the free roaming locomotion patterns of the worms with the speed metric being reported for this study. These assays were established according to previous recorded methods (Amrit et al., 2014; Keith et al., 2014; Mack et al., 2018; Dirksen et al., 2020).

[0102] 2.5 Intestinal permeability assay

[0103] The animals were raised as described previously for lifespan assays. On day 8, the animals were removed from the NGM plates and suspended for 3 h in liquid cultures with blue food dye (FD&C Blue #1, B0790, TCI, 5.0% wt / vol in liquid NGM). The animals were then washed with M9 to remove the unabsorbed dye. Then, the images were captured using a stereo microscope (Nikon S74747) with a D1000 camera for the presence or absence of blue food dye in the body cavity and analyzed using LAS X software (Leica). The following calculation was used to determine the percent of intestinal leakage "permeability":Smart

[0104] Three or more independent experiments were carried out, equaling 8-10 animals per condition. This is as was adapted from the previous methods (Gelino et al., 2016; Kim and Moon, 2019). Data were analyzed using GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, California, United States).

[0105] 2.6 Analysis of stress reporters

[0106] The expression of the stress reporters was measured according to Manjarrez and Mailler, 2020, with supporting evidence for heat shock induction of these stress reporters from An and Blackwell (2003), Bar-Ziv et al. (2020), Bischof et al. (2008), Chen et al. (2023), Labbadia et al. (2017),Taylor et al. (2021), Yoneda et al. (2004). The hsp-4::GFP positive control was treated with tunicamycin for 6 h at 20°C, with a 24-h recovery at 20°C prior to imaging (Yoneda et al., 2004; Bischof et al., 2008). As a hsp-6p::GFP positive control, 1-day-old worms were heat-shocked for 6 h at 30°C, with a 2-h recovery period at 20°C. All experimental measurements were taken under basal conditions: tunicamycin with 50 ng / mL, or heat-shocked at 35°C, for 30 min followed by a 1-h recovery period at 20°C prior to imaging. The images were acquired using a Leica DMi8 fitted with a SpectraX illuminator (Lumencor), an ORCA Flash4.0 v2 sCMOS camera (Hamamatsu), and LAS X software (Leica). Relative fluorescence units (RFUs) were calculated using a LAS X relative fluorescence calculator using a 200 x 200-pm square as a background measurement for the fluorescence intensity of the worm. F(t) = fluorescence channel / region of interest (ROI); F(0) = fluorescence channel / background (Bkg), and K is set to 1 as normalized EGFP (Stepanenko et al., 2008):

[0107] Upregulation of the positive control for each stress reporter was used to obtain the Fmax (maximum reporter intensity) (Manjarrez et al., 2020; Manjarrez and Mail le r, 2020). The normalized values were plotted, and p-values were generated by the nested t-test using GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, California, United States).

[0108] 2.7 Statistics and reproducibility

[0109] Prism 9.5.1 software was used for the survival analysis, using the log-rank (Mantel-Cox) method which analyzed the significance of difference in the overall curve. The Gehan-Breslow- Wilcoxon method was used to assess the significance of survival earlier versus later in the survival timeline. The statistical analysis resulting from the Mantel-Cox, Gehan-Breslow-Wilcoxon, and nested and Student's t-test, in all cases, showed that p < 0.05 was considered significant. An asterisk, in the figures, indicates statistical significance of the aforementioned statistical analysis as compared to its indicated reference. At least three biological replicates comprise all the referenced datasets.

[0110] 3 Results

[0111] 3.1 Survival

[0112] The effect of CeMbio, the laboratory-derived microbiome based on natural isolates, and CeMbio supplemented with L. lactis or L. mesenteroides on the survival of C. elegans was compared to that of the commonly used E. coli, OP50. The results showed that all three CeMbio treatments exhibited significant differences in survival compared to OP50 (FIG. 1). While CeMbio showed the longest median survival and overall lifespan when supplemented with L. lactis or L. mesenteroides, it demonstrated a reduction in the median survival and overall lifespan, contrary to our initial expectation (FIG. 1). This survival analysis indicates that the supplementation of L. lactis or L. mesenteroides to CeMbio had a negative effect on the balance of the differentiated CeMbio microbial community.

[0113] This led to the possibility that supplementation of either L. lactis or L. mesenteroides to the undifferentiated OP50 laboratory strain would improve the lifespan and median survival of the nematodes compared to the OP50 alone, in which both lactic acid bacteria strains and combined OP50 conditions are shown to colonize the C. elegans gut (SI). After investigating the effect of supplementing OP50 with L. lactis or L. mesenteroides, a positive correlation was discovered with the extension of the median and overall lifespan, without showing any signs of developmental arrest associated with either potential probiotic strain (FIG. 2, Panels A-C; FIG. 11). These results indicate that the nutrients / metabolites derived by supplementing L. lactis or L. mesenteroides with OP50 must have advantageous effects by differentiating the C. elegans axenic OP50 strain. Most of the lactic acid bacterial strains or supplementations exhibited extensions in the median and overall lifespan within 13.33%-33.33% and 25%-29%, respectively. The L. / octis-supplemented OP50 or L. lactis monoculture only shows significant differences when analyzed for early death events by the Gehan-Breslow-Wilcoxon test. The log-rank test proved insignificant between the supplementation and the monoculture for L. lactis. However, additional support for the beneficial contribution of nutrients / metabolites of L. mesenteroides intensified with the growth on the monoculture, which exhibits a lifespan extension that exceeds of all biomes tested (FIG. 2, Panel C), with an 87% increase in the median survival and a 67% increase in the overall lifespan beyond the standard OP50. While L. mesenteroides is not known to produce antimicrobials such as nisin, L. lactis has been reported to produce nisin (Khelissa et al., 2021). A significant reduction in the survival rate of C. elegans has been observed with exposure to nisin concentrations higherthan 0.2 mg mL-1 (Boelter et al., 2023). However, since the addition of lactic acid bacteria to OP50 has led to a significant improvement inthe median and overall survival in simulated biomes, the deleterious effect of nisin produced (if any) by L. lactis was not observed.

[0114] 3.2 Morphology and locomotive behavior

[0115] In the OP50 + L.m. group, the nematodes were found to be morphologically distinct, being shorter, thinner, and possessing a smaller area than their counterparts in the OP50 and OP50 + L.l. groups (FIG. 3, Panels A-C; FIG. 12). Additionally, these nematodes displayed slower locomotor behavior compared to those in the OP50, OP50 + L.I., and L.m. groups (FIG. 3, Panel D; FIG. 12). Nematodes in the OP50 + L.l. group were shorter and wider than those in the OP50 group, yet longer and wider than those in the OP50 + L.m. group (FIG. 3, Panels A, B; FIG. 12). They were significantly larger in area and displayed faster locomotion than those in the OP50 + L.m. group, but did not significantly differ from the OP50 group in these aspects (FIG. 3, Panels C, D; FIG. 12). The L.m. group nematodes were shorter, thinner, and smaller than their counterparts in the OP50, OP50 + L.L, and L.l. groups. However, they displayed faster locomotive behavior than the OP50 + L.m., OP50 + L.I., and L.l. groups (FIG. 3, Panels A-D; FIG. 12). In the L.l. group, nematodes were longer, wider, and larger in area than their L.m. counterparts (FIG. 3, Panels A-C; FIG. 12). Interestingly, two distinct widths were observed in this group, with measurements varying around the mean (FIG. 3, Panel B). These nematodes exhibited slower locomotive behaviors than those in the L.m. group (FIG. 3, Panel D).

[0116] 3.3 Progeny production

[0117] In terms of progeny production, the OP50 + L.m. group, despite their reduced speed, produced a higher number of progeny than the L.m. monoculture (FIG. 4, Panel A). The OP50 + L.l. group produced progeny equivalent to those of the OP50 group and at a higher level than those of the L.l. monoculture (FIG. 4, Panel A). The L.l. group produced fewer progeny than both the OP50 and OP50 + L.l. groups (FIG. 4, Panels A, B). The L.m. group showed a decrease in progeny production on the third day of the reproductive cycle compared to the OP50 group and produced fewer total progeny than the OP50 and OP50 + L.m. groups (FIG. 4, Panels A, B). Despite this, the L.m. group continued to produce progeny for a longer duration at a higher level than those of the other groups (FIG. 4, Panel B).

[0118] 3.4 Intestinal permeability

[0119] Assessing intestinal permeability using the Smurf assay revealed an increase in 8-day-old L.m. worms compared to the OP50 group (FIG. 5). Similarly, an increased intestinal permeability wasobserved in the 8-day-old L.I. group, indicating that these longer-lived worms also had increased intestinal permeability akin to the L.m. monoculture group (FIG. 5). However, there was no significant increase in the intestinal permeability that was observed in the OP50 + L.m. or OP50 + L.m. group (FIG. 5).

[0120] In the context of reactive oxygen species (ROS) stress responses, the data showed that the basal and heat shock (HS) levels in the OP50 + L.m. group were elevated compared to those in the L.m. group (FIGS. 6 and 13). Despite this increase, the basal and HS levels remained relatively unchanged upon extrinsic heat shock insults. In the OP50 + L.l. HS group, the ROS stress response was found to be elevated compared to that in the L.l. HS group. However, similar to the OP50 + L.m. group, the basal and HS levels remained relatively unchanged upon insults (FIGS. 6 and 13). In the L.m. group, the basal ROS levels were found to be below those in the OP50 and OP50 + L.m. group, as well as the HS groups for these culture groups (FIGS. 6 and 13). The basal and HS ROS response levels in the L.m. group remained relatively unchanged upon stress insults as measured by the gcs- 1 reporter strain, indicating the lowest measured stress levels (FIGS. 6 and 13). The ROS stress response in the L.l. HS group was found to be below that of the OP50 HS and OP50 + L.l. HS group (FIGS. 6 and 13). However, the basal group showed a slight increase over the HS group but was otherwise unchanged upon insult (FIGS. 6 and 13).

[0121] 3.6 Unfolded protein response (UPRcyt) stress response

[0122] Concerning the UPRcyt stress responses, interesting patterns were observed across different groups. In the OP50 + L.m. group, the basal stress response levels were significantly higher than those in the OP50 and L.m. group. However, these levels were decreased in comparison to the OP50 + L.m. HS and L.m. HS response (FIGS. 7 and 14). For the OP50 + L.l. group, the basal stress response levels were notably decreased compared to both the OP50 + L.l. HS and L.l. groups (FIGS. 7 and 14). Thus, the OP50 + L.l. group displayed a reduced UPRcyt basal stress response. In the L.m. group, the UPRcyt basal stress response was significantly decreased compared to the OP50, OP50 + L.m., and L.l. group (FIGS. 7 and 14). However, the L.m. HS stress response in L.m. showed a robust increase over basal L.m. levels, with OP50 + L.m. HS, and L.l. HS, indicating an elevated UPRcyt stress response upon heat shock in the L.m. group. Lastly, in the L.l. group, the UPRcyt basal level was decreased relative to both the L.l. HS, OP50, and OP50 + L.l. groups (FIGS. 7 and 14). The L.l. HS stress response decreased compared to the OP50 HS and L.m. HS stress responses, indicating a lowered UPRcyt response upon heat shock in the L.l. group.

[0123] 3.7 Unfolded protein response (UPRER) stress response

[0124] In terms of UPRER stress responses, short-term treatment with tunicamycin showed only marginal increases although not significant for OP50, OP50 + L.m., and L.l. While, showing decreases with short-term exposure for OP50 + L.l. and L.m. compared to controls (FIG. 8, Panel A; FIG. 15, Panel A). OP50 + L.l. did show an increase amount of basal stress over OP50 + L.m. under DMSO treatment but did not show significant differences upon tunicamycin treatment (FIG. 8, Panel A). However, the OP50 tunicamycin treated group showed a significant increase in stress over the L.m. treated group upon short-term exposure (FIG. 8, Panel A; FIG. 15, Panel A). The other possible induction of the hsp-4 transgene according to the CGC, heat shock, shows a diverse trend across the different groups studied. For the OP50 + L.m. group, the UPRER basal stress response levels were found to be elevated above those in the OP50 + L.l. group. A slight elevation in the HS to basal level was observed, although the changes in these levels upon extrinsic heat shock insults remained relatively unchanged (FIG. 8, Panel B; FIG. 15, Panel B). In the OP50 + L.l. group, the levels in the UPRER HS group were significantly elevated compared to the basal response levels in the same group. These levels were also found to be decreased relative to the OP50 and OP50 HS groups. Furthermore, the OP50 + L.l. response level decreased compared to the OP50 + L.m. response level (FIG. 8, Panel B; FIG. 15, Panel B). In the L.m. group, the UPRER basal and L.m. HS stress responses remained relatively unchanged upon insult, indicating that this group had robust resistance to UPRER stress (FIG. 8, Panel B; FIG. 15, Panel B). No significant differences were observed between these responses and those of the other groups. In the L.l. group, the UPRER HS group was found to decrease relative to the OP50 HS group, indicating a reduced response upon heat shock in the L.l. group. Interestingly, the basal groups showed a slight increase over the HS groups or slight increase or decreases in most groups with tunicamycin treatment, however; these changes in most treatments were not significant, and the responses were otherwise relatively unchanged (FIG. 8, Panels A, B; FIG. 15, Panels A, B).

[0125] 3.8 Unfolded protein response (UPRmt) stress response

[0126] The UPRmt stress responses across various groups demonstrated intriguing patterns. In the OP50 + L.m. group, the HS response showed a marked decrease compared to the OP50 HS and OP50 + L.l. HS responses (FIGS. 9 and 16). However, under these conditions, the basal level of response was slightly elevated over the OP50 + L.m. HS levels, but this elevation was not significant (FIGS. 9 and 16). In the context of the OP50 + L.l. group, the level of the UPRmt HS group waselevated compared to the basal response level (FIGS. 9 and 16). While the level of the OP50 + L.l. HS group was increased above the level of the OP50 + L.m. HS group, there were no significant differences between OPSO HS or L.l. HS groups (FIGS. 9 and 16). Investigating the L.m. group, the UPRmt showed that the L.m. basal and L.m. HS stress responses were relatively unchanged upon insults, indicating the lowest measured stress levels. Basal and HS stress responses in L.m. were found to be decreased below those in the OP50 and OP50 HS groups (FIGS. 9 and 16). Moreover, L.m. HS stress response levels decreased relative to those in the L.l. HS group (FIGS. 9 and 16). In the L.l. group, the UPRmt showed a slight increase in the HS response over the basal condition upon insult, but this was not significant (FIGS. 9 and 16). However, the L.l. HS group exhibited an increased response compared to the L.m. HS group, while maintaining an overall higher response in the basal and HS level (FIGS. 9 and 16).

[0127] 4 Discussion

[0128] The use of probiotics, particularly lactic acid bacteria, has been increasingly studied for its potential health benefits. One of the most promising areas of research has been in its impact on the lifespan, stress response, and nutrient uptake. Several studies have shown that probiotics can lead to a longer lifespan in various organisms, including C. elegans. In addition to the impact of probiotics on the lifespan, they have also been found to regulate stress responses more tightly. For instance, a study published in PNAS in 2011 found that the probiotic Lactobacillus reuteri reduced stress-induced cortisol levels in mice (Bravo et aL, 2011). This indicates that the introduction of probiotics can help individuals better manage stress, which is essential for their overall health and wellbeing.

[0129] The current study demonstrates that significantly lower basal and stress levels compared to controls are indicative of a positive early health response. These findings were consistent across different cultured groups, which were most significant in the L. mesenteroides monoculture, emphasizing the potential probiotic applicability of these biomarkers in predicting early health responses in the C. elegans model system (Dhama et al., 2019). The relationship between L. mesenteroides' stress response and survival probability reveals that a balanced and tightly modulated stress response is associated with the longest survival probability (Vermeulen and Loeschcke, 2007). Upon extrinsic stress insults, L. mesenteroides showed a capacity to buffer stress with relatively unchanged stress responses and was associated with a significantly higher survival probability compared to those with the most altered basal-to-stress responses, OP50 + L.l. Thisindicates the accumulation of protective compounds in the L. mesenteroides group, consistent with the uptake of additional advantageous nutrients from this probiotic supplement (Miyamoto et al., 2023).

[0130] The analysis of stress response-related biomarkers revealed that L. mesenteroides, with a stable stress response, exhibited minimal fluctuations in the levels of the ROS, UPRER, and UPRmt response genes, indicating an efficient adaptation mechanism to maintain homeostasis (Grompone et al., 2012; Park et al., 2018; Dhama et al., 2019; Kumar et al., 2022; Yun et al., 2022; Zhang et al., 2022). This well-regulated stress response could play a pivotal role in boosting the organism's resilience and ability to cope with insults, ultimately leading to increased survival probabilities provided through probiotic supplementation with the L. mesenteroides culture. Although this was seen most prevalently in the L. mesenteroides monoculture, the supplementation of the OP50 axenic culture with L. mesenteroides, OP50 + L.m., also showed a similar trend, only not to the same degree (Miyamoto et aL, 2023).

[0131] Furthermore, the study revealed that L. mesenteroides with relatively unchanged stress responses exhibited better overall health span parameters, including anatomically beneficial features, which contribute to their longer survival probability and ability to maintain stability during a stress response. However, contradictory to current findings, L. mesenteroides and L. lactis show increased intestinal permeability, even with increased median and overall survival probabilities (Gelino et al., 2016; Kim and Moon, 2019). Even though intestinal permeability has been shown to be associated with irritable bowel syndrome, obesity, chronic kidney disease, and cardiovascular diseases, it shows a link between increased permeability and the promotion of dysbacteriosis (Inczefi et al., 2022). However, another concept is that probiotics can lead to a more permeable intestine, which can lead to more efficient nutrient uptake. This is owing to the fact that probiotics can promote the growth of beneficial bacteria in the gut, which can aid in digestion and nutrient absorption (O'Toole and Jeffery, 2015; Requena et aL, 2018; Annapure and Nair, 2022). In turn, this can lead to improved overall health and increased energy levels. As such, the maintenance of a physiological balance in the face of stressors could be a key feature determining the longevity and overall health of an animal provided with probiotic supplementation (Grompone et al., 2012; Oh et al., 2015; Park et al., 2018; Kumar et al., 2022; Yun et al., 2022; Zhang et al., 2022).

[0132] In conclusion, the results of this study underscore the importance of a balanced and tightly controlled stress response for ensuring the longest survival probability upon oxidative andproteostatic insult. The ability to maintain homeostasis and efficiently buffer extrinsic stressors appears to be a critical determinant of an organism's resilience and survival. Moreover, the efficacy of probiotics can vary depending on the individual's gut microbiome and health status. The use of probiotics has shown promising results for improving the lifespan, stress response, and nutrient uptake. Consequently, the incorporation of probiotics into one's diet or taking probiotic supplements may provide significant health benefits. Despite the promising findings using these potential probiotics, further research is still needed to fully elucidate the molecular mechanisms underlying this phenomenon and to explore potential therapeutic strategies to enhance stress response regulation and improve survival outcomes through the probiotic application of L. lactis and L. mesenteroides.

[0133] 4.1 Summary

[0134] This study highlights the importance of significantly lower basal and stress levels as indicators of an early health response in the C. elegans model system. The results emphasize the potential probiotic applicability of these biomarkers for predicting early health responses, whereas a balanced and tightly modulated stress response was found to be associated with the longest survival probability, which demonstrated significantly longer survival rates than those with altered stress responses. The efficient adaptation mechanisms that maintain homeostasis ultimately lead to an increased survival probability. The relatively unchanged stress responses exhibited better overall health span parameters, contributing to the ability to maintain physiologically balanced condition in the face of stressors, which is a key feature in determining the longevity and overall health of an organism provided with L. lactis and L. mesenteroides as a therapeutic probiotic supplement.

[0135] Supplemental Protocols:

[0136] Developmental Transition:

[0137] To assess the influence the effects of L. lactis and L. mesenteroides and combined OP50 on the developmental transition of wild-type C. elegans, nematodes were raised on monocultures and combined lawns. Worms were synchronized through an egg lay protocol on their respective bacterial lawn. The number of adults transition from L4 to young adult (through the appearance of a vulva) over time were scored on an hourly basis at 56-65 hours post egg lay (Dirksen et al. 2020).

[0138] Colonization Assay

[0139] Modified from Dirksen et al. 2020, we adapted the colonization assay to determine if there was growth from the monocultures extracted from the gut of the egg lay synchronized worms at the 1-day old adult stage. We counted the lactic acid bacterial colonies after growing overnight at 32°C to determine the CFU values for colonization of the 30 - 1-day old C. elegans. The protocol that was largely followed was previously published from Zhang et al. 2020 and is available online on protocols. io (DOI: dx.doi.org / 10.17504 / protocols.io.rtzd6p6) (Zhang et al. 2020; Dirksen et al. 2020).

[0140] References:

[0141] Dirksen, Philipp, Adrien Assie, Johannes Zimmermann, Fan Zhang, Adina-Malin Tietje, Sarah Arnaud Marsh, Marie-Anne Felix, et al. 2020. "CeMbio - The Caenorhabditis Elegans Microbiome Resource." G3 (Bethesda, Md.) 10 (9): 3025-39.

[0142] Zhang, Fan, Jessica L. Weckhorst, Adrien Assie, Anastasia S. Khodakova, Mario Loeza- Cabrera, Daniela Vidal, and Buck S. Samuel. 2020. "High-Throughput Assessment of Changes in the Caenorhabditis Elegans Gut Microbiome." Methods in Molecular Biology 2144: 131-44.Table 1Table 2Example 2

[0143] In this Example, an acid tolerance test of the probiotic compositions constructed in accordance with the present disclosure was performed. In particular, the acid tolerance of LAB L. mesenteroides growth in MRS adjusted to pH 3.0 with HCI was tested.

[0144] 1 ml of overnight LAB culture was inoculated into 9 mL of acidified MRS (pH 3.0) and then incubate at 37°C for 3 hours. Samples were plated at intervals (0, 2, & 3 hours) to determine CFU, for acid tolerance. A control sample was included by inoculating 1 mL of the overnight LAB culture into 9 mL only MRS broth to ensure growth of strain. The experiment was conducted in triplicate, and 400 pl of dilution was plated.

[0145] CFU / ml was calculated as Volume plates (mL) number of colonies x Dilution factor. 0- hour dilution factor 1,000,000; 2-hour dilution factor 10; 3-hour dilution factor 10. 400 pl was plated for both time points, and this was repeated in triplicate.

[0146] The results are shown in FIG. 17.

[0147] The control at 0 HR was 2.29 X 108CFU / ml -> log (2.29 X 108) = 8.36. With MRS reduction to pH 3.0 after 2-HR was 1.28 X 105CFU / ml -> log™ (1.28 X 105) = 5.11. With MRS reduction to pH 3.0 after 3-HR was 2.03 X 104CFU / ml -> log (2.03 X 104) = 4.31.

[0148] These results demonstrate that the strain of Leuconostoc mesenteroides used in the present disclosure, when exposed to medium at a pH of 3.0, resulted in a 3-log reduction in viable cells compared to the control after two hours, and further resulted in a 3.8-log reduction after three hours.Example 3

[0149] In this Example, a bile tolerance test of the probiotic compositions constructed in accordance with the present disclosure was performed. In particular, the bile tolerance of LAB L. mesenteroides to bile at 0.3% was tested.

[0150] 1 ml of overnight LAB culture was inoculated into 9 mL of MRS with 0.3% bile salts and incubated at 37°C for 24 hours.TABLE 3

[0151] Samples were plated at 0 hours and 24 hours after inoculation into the bile media at the dilutions indicated in Table 3. The conclusion is reported in CFU / ml after the 24-hour growth / incubation period, as shown in FIG. 18.

[0152] Exports from iBright colony count analysis. GraphPad Prism statistical analysis of CFU / mL survival. CFU / mL=Volume plated (mL)Number of coloniesxDilution factor. 0-hour dilution factor 100,000; 24-hour dilution factor 10,000. 400 pL was plated for both time points. Repeated in triplicate.

[0153] As shown in FIG. 18, the control at 0 HR was 3.60 X 107CFU / ml --> logw (3.60 X 107) = 7.56. With 0.3% bile after 24 HR was 1.88 X 106CFU / ml --> logw (1.88 X 106) = 6.27.

[0154] These results demonstrate that the strain of Leuconostoc mesenteroides used in the present disclosure, when exposed to 0.3% bile, resulted in a 1.28 log reduction in viable cells compared to the control after 24 hours.Example 4

[0155] In this Example, a hemolysis assay of the probiotic compositions constructed in accordance with the present disclosure was performed.

[0156] It is known in the art that some enterotoxins present in certain bacteria cause hemolysis. Therefore, this Example utilized an assay to detect if any hemolytic enterotoxins were present in the LAB L. mesenteroides of the present disclosure, and if so, what type(s) of hemolytic enterotoxin(s) is / are present in this strain.

[0157] I n this Example, supernatant was inoculated on blood agar plates, and the plates were examined for zones of hemolysis (3-hemolysis indicates complete lysis).

[0158] H emolytic bacterial activity was tested by dropping 2 pl of an overnight culture of the LAB L. mesenteroides or P. aeruginosa PA14 onto a blood agar plate (three plates for LAB and two plates for PA14). After 48 hours of incubation at 37°C, hemolysis was evident by a greenish or yellowish circle. The assay was checked at 24 hours and completed at 48 hours.

[0159] FIG. 19 contains photographs of the blood agar plates after 48 hours. As can be seen on the left side of FIG. 19, the PA14 plates were greenish-yellow with full clearing of the agar, thus indicating beta hemolysis (3-hemolysis). In contrast, as seen on the right side of FIG. 19, the L. mesenteroides plates were greenish-yellow without full clearing of the agar, thus demonstrating alpha hemolysis (a-hemolysis).

[0160] In summary, the control PA14 exhibited the known (3-hemolysis, while the L. mesenteroides of the present disclosure exhibited a-hemolysis. This is due to the presence of a- hemolysin, which was confirmed by genome-wide sequencing (GWS; see Example 6 below).Example 5

[0161] In this Example, an antibiotic resistance assay of the probiotic compositions constructed in accordance with the present disclosure was performed. In particular, the resistance of LAB L. mesenteroides to various antibiotics was tested.

[0162] This experiment began with active broth cultures for LAB in MRS medium and P. aeruginosa in LB (must be actively growing cultures). The active broth cultures were diluted in a 0.9% saline to a 625 nm absorbance of ~0.19, and this dilution was used within 15-20 minutes. Using the diluted broth cultures, bacterial lawns were prepared using the chosen organism on Mueller Hinton agar plates (pH 7.2-7.4). This was repeated with as many agar plates as necessary, depending on the number of antibiotic disks and the size of the petri dish (100 x 15 = 8 discs / plate; 150 x 15 = 12 discs / plate). P. aeruginosa PA14 was used as a control strain, as shown in Table 4.TABLE 4

[0163] The lids were left open for an amount of time sufficient to allow the plates to dry, as the surface needs to be dry before moving on to the next step. The lids were typically left open for 3-5 minutes, but not longer than 15 minutes. Then using forceps, the discs were placed at an equal distance radial pattern with a single disc in the center. It was ensured that notations were made about the identity of each antibiotic chosen, as well as its concentration given, and the location on the plate. The disc was then gently pressed onto the agar to complete the placement. The forceps were then cleaned with ethanol after placing each disc, and the discs were not moved once they touched the agar plate. Templates for the placement of the discs on the 100 mm and 150 mm plates were provided.

[0164] The plates were inverted and incubated at 35°C for 24 and 48 hours, for the LABs of the present disclosure. This incubation period could be shorter for other bacterial strains, such as P. aeruginosa, where 18 and 24 hours was used. The regions were observed for no growth at 24 and 48 hours (or 18 and 24 hours for P. aeruginosa).

[0165] Two different methods for measuring were utilized. First, from the bottom of the plate, the edge of the disc to the edge of the growth was measured with a millimeter ruler across the middle of the disc from edge to edge. However, if the region from another disc was overlapping, the measurement was taken from the center of the disc out to the non-overlapping edge, and that measurement was doubled. Then Table 5 was utilized to determine if the organism is Susceptible (S), Intermediate (I), or Resistant to each antibiotic by usingthe reference ranges. This Table for zone of inhibition measurements for common antibiotics is based on the Clinical and Laboratory Standards Institute (CLSI).TABLE 5

[0166] Finally, results of inhibition zone measurement tables were prepared that list S / l / R in the results along with the measurements. See Table 6 for the results obtained with L. mesenteroides and Table 7 for the results obtained with PA14.TABLE 6TABLE 7

[0167] PA14 grew within the first 18-24 hours as expected, while the LAB took 48 hours as projected to measure in this assay. PA14 cultures were diluted 1:10 in 0.9% saline. LAB cultureswere not diluted, as their growth progression is low in this assay. This was completed for L.m. and L.l. x3 on MRS plates with one MH PA14 plate with the original orientation as a control for the antibiotics. The LAB bacteria did not grow well enough to assay this resistance on MH plates; therefore, the need to use MRS plates to assess the qualities of these bacterial strains was necessitated. The LAB bacterial strains were grown on MRS overnight at 35°C, and the PA14 on MH were grown overnight at 37°C. Then will be assessed as above for antibiotic resistance.

[0168] PA14 was used as the control strain It showed resistance to Ampicillin, Amoxicillin / Clavulanic Acid, Bacitracin, Erythromycin, Kanamycin, Novobiocin, Oxacillin, Penicillin G, Vancomycin, and Sulfamethoxazole / Trimethoprim. PA14 also showed an intermediate response to Chloramphenicol and Streptomycin. In addition, PA14 showed susceptibility to Ciprofloxacin, Neomycin, and Tetracycline.

[0169] The Leuconostoc mesenteroides strain of the present disclosure showed resistance to Ciprofloxacin, Kanamycin, Oxacillin, and Streptomycin. The L. mesenteroides strain also showed an intermediate response to Neomycin, Novobiocin, and Penicillin G. In addition, the L. mesenteroides strain showed susceptibility to Ampicillin, Amoxicillin / Clavulanic Acid, Bacitracin, Chloramphenicol, Erythromycin, and Tetracycline.

[0170] These results indicate that probiotic compositions containing the L. mesenteroides strain of the present disclosure would be particularly useful for simultaneous or sequential administration to a subject receiving antibiotic treatment with at least one of Ciprofloxacin, Kanamycin, Oxacillin, and Streptomycin. The probiotic compositions containing the L. mesenteroides strain of the present disclosure would also be useful for simultaneous or sequential administration to a subject receiving antibiotic treatment with at least one of Neomycin, Novobiocin, and Penicillin G.Example 6

[0171] A genomic sequence of the strain of L. mesenteroides isolated in Example 1 has been obtained. Various samples of the genomic sequence are provided and have been assigned SEQ ID NOS:l-6. In particular, the genome of L. mesenteroides is carried on two plasmids; SEQ ID NOS:1, 3, and 5 are samples of the sequence of the first plasmid, whereas SEQ ID NOS:2, 4, and 6 are samples of the sequence of the second plasmid. Thus, SEQ ID NOS:l-2 form the genomic sequence of a first sample (L_m-1); SEQ ID NOS:3-4 form the genomic sequence of a second sample (L_m-2); and SEQ ID NOS:5-6 form the genomic sequence of a third sample (L_m-3).

[0172] Various samples of the genomic sequence of L. mesenteroides isolated in Example 1 were compared to the reference genome of Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293, Reference Genome ASM 1444vl, NCBI Taxonomy ID: 203120, that was isolated from fermenting olives (i.e., a fruit source). A total of 37,540 MNPs were identified, which were (12,531, 12,560, and 12,449) across the three samples, with an average of 12,513 MNPs per sample. A total of 75,663 SNPs were identified, which were (25,183, 25,183, and 25,297) across the three samples, with an average of 25,221 SNPs per sample. A total of 4,414 indels were identified, which were (1,466, 1,495, and 1,453) across the three samples, with an average of 1,471 indels per sample. A total of 1,544 other variants were identified, which were (505, 517, and 522) across the three samples, with an average of 515 other variants per sample. On average, each sample maintained a 1.623 ts / tv ratio.

[0173] Average Nucleotide Identity: A BLAST-free, ANIb-like workflow was utilized. The query genome was tiled into non-overlapping 1,020-bp fragments. For each fragment, canonical 21-mer seeding (~255-bp stride) was performed against a k-mer index of the reference (both strands), candidate 1,020-bp windows (±50-100 bp offsets) were selected, and the best window was chosen by ungapped identity. On a stratified, genome-wide subsample (~60 fragments), Smith-Waterman local alignment (band 20; scoring ±2 / -3 / -4) was run to recover indels. Gapped ANI is the mean gapped identity across the subsample; coverage is the fraction of all fragments that mapped. Results are shown in Table 8.TABLE 8

[0174] Based on the results shown in Table 8, and using the >95-96% gapped ANI guideline, the L_m-2 and L_m-3 samples isolated from organic basil of the present disclosure are demonstrated to be the same species as the reference genome of Leuconostoc mesenteroides ATCC 8293. In addition, the L_m-2 and L_m-3 samples of the present disclosure are near-clonal (i.e., the same strain). Further, the assembly of the reference genome ATCC 8293 versus the L_m- 2 and L_m-3 samples of the present disclosure reflects that the L. mesenteroides of the presentdisclosure is a different strain from the reference genome ATCC 8293 and possesses at least about 12% difference in sequence from the reference genome.

[0175] Therefore, the results of Table 8 demonstrate that the L. mesenteroides of the present disclosure that is isolated from a vegetable source possesses substantial structural differences (i.e., at least about 12% difference in genomic sequence) from the reference L. mesenteroides strain isolated from a fruit source.Example 7

[0176] A genomic sequence of the strain of L. lactis isolated in Example 1 has also been obtained. The L. lactis genome is assembled as one plasmid. Various samples of the genomic sequence are provided and have been assigned SEQ ID NOS: 7-12.

[0177] The Lactococcus lactis subsp. lactis ATCC 19435, NCBI / DDBJ accession: AB008215 - 16S rRNA gene, widely used for species-level identification, was used for positive identification of the species / subspecies reference in the genome sequence of the Lactococcus lactis strain of the present disclosure.

[0178] It is well-known that Lactococcus is a diverse species. When a test strain differs significantly from the reference strain, many reads will not map correctly. Consequently, there are genetically distinct differences between Lactococcus strains adapted to dairy and plant environments. Notably, wild strains exhibit a greater diversity compared to typical dairy strains.

[0179] One notable consequence of strain diversity is that attempting to map with more closely related genomes increases the likelihood of success.

[0180] To identify a suitable candidate, an ANI search was conducted using SEQ ID NOS:10- 12. Although mapping was still unsuccessful due to the aforementioned reasons, the ANI did indicate that the coverage was most consistent with one source genome from a frozen pea (vegetable source). Reference genome ASM47825v2 (NCDO 2118) showed a coverage of 84.71% and a gapped ANI of 83.01%. In contrast, those from the Cultivarium collection ASM3384296vl (ATCC 19435) or used in the cultivation of soft cheese ASM686vl (IL1403) had a gapped ANI of 70.72 at 0.31% coverage and 32.16 at 0.2% coverage, respectively. The closely related L. cermoris ASM942vl was absent in coverage and ANI. Therefore, this closely often overlapping species with lactis was ruled out.NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0181] Illustrative embodiment 1. A probiotic composition, comprising an effective amount of at least one therapeutic bacterium or a spore thereof, wherein the at least one therapeutic bacterium or spore thereof comprises a strain of lactic acid bacterium or a spore thereof that is isolated from a vegetable source.

[0182] Illustrative embodiment 2. The probiotic composition of Illustrative embodiment 1, wherein the probiotic composition comprises at least two lactic acid bacteria or spores thereof.

[0183] Illustrative embodiment 3. The probiotic composition of Illustrative embodiment 1 or 2, wherein the probiotic composition comprises at least one of Lactococcus lactis (L. lactis) and Leuconostoc mesenteroides (L. mesenteroides).

[0184] Illustrative embodiment 4. The probiotic composition of Illustrative embodiment 3, wherein L. mesenteroides has a genomic sequence comprising at least a portion of at least one of SEQ ID NOS:l-6, and L. lactis has a genomic sequence comprising at least a portion of at least one of SEQ ID NOS:7-12.

[0185] Illustrative embodiment 4A. The probiotic composition of Illustrative embodiment 3, wherein L. mesenteroides has a genomic sequence that is at least about 90% identical to at least a portion or all of at least one of SEQ ID NOS:l-6, and / or L. lactis has a genomic sequence that is at least about 90% identical to at least a portion or all of at least one of SEQ ID NOS:7-12.

[0186] Illustrative embodiment 4B. The probiotic composition of Illustrative embodiment 3, wherein L. mesenteroides has a genomic sequence that is at least about 91%, about 92%, or about 93% identical to at least a portion or all of at least one of SEQ ID NOS:l-6, and / or L. lactis has a genomic sequence that is at least about 91%, about 92%, or about 93% identical to at least a portion or all of at least one of SEQ ID NOS:7-12.

[0187] Illustrative embodiment 4C. The probiotic composition of Illustrative embodiment 3, wherein L. mesenteroides has a genomic sequence that is at least about 94%, about 95%, or about 96% identical to at least a portion or all of at least one of SEQ ID NOS:l-6, and / or L. lactis has a genomic sequence that is at least about 94%, about 95%, or about 96% identical to at least a portion or all of at least one of SEQ ID NOS:7-12.

[0188] Illustrative embodiment 4D. The probiotic composition of Illustrative embodiment 3, wherein L. mesenteroides has a genomic sequence that is at least about 97%, about 98%, or about 99% identical to at least a portion or all of at least one of SEQ ID NOS:l-6, and / or L. lactis has a genomic sequence that is at least about 97%, about 98%, or about 99% identical to at least a portion or all of at least one of SEQ ID NOS:7-12.

[0189] Illustrative embodiment 4E. The probiotic composition of any of Illustrative embodiments 1-4D, wherein the strain of lactic acid bacterium or a spore thereof comprises an a-hemolysin.

[0190] Illustrative embodiment 5. The probiotic composition of Illustrative embodiment 3 or 4, wherein the probiotic composition comprises L. lactis and L. mesenteroides.

[0191] Illustrative embodiment 6. The probiotic composition of Illustrative embodiment 5, wherein L. mesenteroides and L. lactis are present in the probiotic composition at a weight ratio in a range of from about 10:1 to about 1:1.

[0192] Illustrative embodiment 7. The probiotic composition of Illustrative embodiment 5 or 6, wherein L. mesenteroides and L. lactis are present in the probiotic composition at a weight ratio in a range of from about 6:1 to about 2:1.

[0193] Illustrative embodiment 8. The probiotic composition of any of Illustrative embodiments 5-7, wherein L. mesenteroides and L. lactis are present in the probiotic composition at a weight ratio in a range of from about 5:1 to about 3:1.

[0194] Illustrative embodiment 9. The probiotic composition of any of Illustrative embodiments 5-8, wherein L. mesenteroides and L. lactis are present in the probiotic composition at a weight ratio of about 4:1.

[0195] Illustrative embodiment 10. The probiotic composition of any of Illustrative embodiments 1-9, wherein the at least one lactic acid bacterium or spore thereof is isolated from an organic herb.

[0196] Illustrative embodiment 11. The probiotic composition of any of Illustrative embodiments 1-10, wherein the at least one lactic acid bacterium or spore thereof is isolated from basil.

[0197] Illustrative embodiment 11A. The probiotic composition of any of Illustrative embodiments 1-11, wherein the at least one lactic acid bacterium or spore thereof exhibits substantial acid tolerance at a pH greater than or equal to about 3.

[0198] Illustrative embodiment 11B. The probiotic composition of any of Illustrative embodiments 1-11A, wherein the at least one lactic acid bacterium or spore thereof exhibits substantial bile tolerance at a bile concentration of at least about 0.3% (w / w).

[0199] Illustrative embodiment 12. The probiotic composition of any of Illustrative embodiments 1-11, further comprising at least one pharmaceutically acceptable carrier or excipient.

[0200] Illustrative embodiment 13. The probiotic composition of Illustrative embodiment 12, wherein the pharmaceutically acceptable carrier or excipient is selected from the group consisting of hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose acetate succinate, microcrystalline cellulose, milk powder, trehalose, xanthan gum, banana powder, dextran, maltose, lactose, glutamic acid, glycerin, PBS, mannitol, partially pregelatinized starch, silicon dioxide, magnesium stearate, maltodextrin, functionalized mineral (Omya), Direct compression grade mannitol, polyvinylpyrrolidone (PVP), dibasic calcium phosphate (DCP), and combinations thereof.

[0201] Illustrative embodiment 14. The probiotic composition of any of Illustrative embodiments 1-13, wherein the strain of lactic acid bacterium or a spore thereof has been lyophilized.

[0202] Illustrative embodiment 15. The probiotic composition of any of Illustrative embodiments 1-14, wherein the strain of lactic acid bacterium or a spore thereof is encapsulated.

[0203] Illustrative embodiment 16. The probiotic composition of Illustrative embodiment 15, wherein the encapsulation method is selected from at least one of the following: microencapsulation (such as for use in chocolate, bakery items, etc.), polymeric encapsulation, spray drying, emulsion, extrusion, spray freeze drying, layer by layer, ionic gelation, vibration technology, sol-gel encapsulation, cyclodextrin inclusion, solvent evaporation method, and combinations thereof.

[0204] Illustrative embodiment 17. The probiotic composition of any of Illustrative embodiments 1-16, further comprising at least one prebiotic.

[0205] Illustrative embodiment 17A. The probiotic composition of Illustrative embodiment 17, wherein the at least one prebiotic is selected from the group consisting of 3-glucan, galactooligosaccharides, fructo-oligosaccharides, starch, trans-galacto-oligosaccharides, glucooligosaccharides, glyco-oligosccharides, lactulose, lactitol, malto-oligosaccharides, xylooligosaccharides, stachyose, raffinose, inulin, isomalto-oligosaccharides, xylo-oligosaccharides, lacticol, lactulose, cereal fibre, short-chain oligosaccharide, and combinations thereof.

[0206] Illustrative embodiment 18. The probiotic composition of any of Illustrative embodiments 1-17, wherein the probiotic composition is formulated for oral administration.

[0207] Illustrative embodiment 19. The probiotic composition of any of Illustrative embodiments 1-18, wherein the probiotic composition is in a form selected from the group consisting of a liquid (i.e., drops), a suspension, an emulsion, a dried powder, a capsule, a foodproduct, a medical food, a chewable, a gummy, a spray-dried nasal product, a sachet, or a combination thereof.

[0208] Illustrative embodiment 20. The probiotic composition of Illustrative embodiment 19, wherein the food product is selected from the group consisting of a yogurt, a kimchi, a dairy product, a fermented food, a yakult drink, a cheese, a chocolate, a bakery item, and combinations thereof.

[0209] Illustrative embodiment 21. The probiotic composition of any of Illustrative embodiments 1-20, wherein the at least one therapeutic bacterium or spore thereof of the probiotic composition is at least partially resistant to at least one antibiotic.

[0210] Illustrative embodiment 21A. The probiotic composition of Illustrative embodiment 21, wherein the at least one antibiotic is selected from the group consisting of Neomycin, Novobiocin, Penicillin G, Ciprofloxacin, Kanamycin, Oxacillin, Streptomycin, and combinations thereof.

[0211] Illustrative embodiment 21B. The probiotic composition of any of Illustrative embodiments 1-21A, wherein the at least one therapeutic bacterium or spore thereof of the probiotic composition is substantially resistant to at least one antibiotic.

[0212] Illustrative embodiment 21C. The probiotic composition of Illustrative embodiment 21B, wherein the at least one antibiotic is selected from the group consisting of Ciprofloxacin, Kanamycin, Oxacillin, Streptomycin, and combinations thereof.

[0213] Illustrative embodiment 22. A method, comprising the step of administering the probiotic composition of any of Illustrative embodiments 1-21C to a subject in need of treatment.

[0214] Illustrative embodiment 23. The method of claim 22, wherein the subject is a mammal.

[0215] Illustrative embodiment 24. The method of Illustrative embodiment 22 or 23, wherein the subject is a human.

[0216] Illustrative embodiment 25. The method of Illustrative embodiment 22 or 23, wherein the subject is a domestic animal.

[0217] Illustrative embodiment 26. The method of any of Illustrative embodiments 22-25, wherein the probiotic composition is orally administered to the subject.

[0218] Illustrative embodiment 27. The method of any of Illustrative embodiments 22-26, wherein the subject is experiencing at least one gastrointestinal (Gl) symptom.

[0219] Illustrative embodiment 28. The method of Illustrative embodiment 27, wherein the at least one gastrointestinal symptom is selected from the group consisting of upset stomach, heartburn, nausea, vomiting, loose bowel movements, and combinations thereof.

[0220] Illustrative embodiment 29. The method of any of Illustrative embodiments 22-28, wherein the subject has an abnormal gut biome.

[0221] Illustrative embodiment 30. The method of Illustrative embodiment 29, wherein administration of at least one antibiotic to the subject has contributed to the abnormal gut biome.

[0222] Illustrative embodiment 31. The method of any of Illustrative embodiments 22-30, wherein the subject is immunocompromised.

[0223] Illustrative embodiment 32. The method of any of Illustrative embodiments 22-31, wherein the subject has cancer.

[0224] Illustrative embodiment 33. The method of any of Illustrative embodiments 22-32, wherein the subject has received at least one chemotherapy and / or radiation treatment, and optionally wherein the at least one chemotherapy and / or radiation treatment is known or suspected to affect the gut biome.

[0225] Illustrative embodiment 34. The method of any of Illustrative embodiments 22-33, wherein the subject has an autoimmune disease.

[0226] Illustrative embodiment 35. The method of any of Illustrative embodiments 22-34, wherein the subject has received at least one immunotherapy treatment, and optionally wherein the at least one immunotherapy treatment is known or suspected to affect the gut biome.

[0227] Illustrative embodiment 36. The method of any of Illustrative embodiments 22-35, wherein the subject has at least one gastrointestinal tract disease or condition.

[0228] Illustrative embodiment 37. The method of any of Illustrative embodiments 22-36, further comprising the step of administering at least one antibiotic to the subject, wherein the at least one therapeutic bacterium or spore thereof of the probiotic composition is at least partially resistant to the at least one antibiotic.

[0229] Illustrative embodiment 37A. The method of Illustrative embodiment 37, wherein the at least one antibiotic is selected from the group consisting of Neomycin, Novobiocin, Penicillin G, Ciprofloxacin, Kanamycin, Oxacillin, Streptomycin, and combinations thereof.

[0230] Illustrative embodiment 38. The method of Illustrative embodiment 37, wherein the at least one therapeutic bacterium or spore thereof of the probiotic composition is substantially resistant to the at least one antibiotic.

[0231] Illustrative embodiment 38A. The probiotic composition of Illustrative embodiment 38, wherein the at least one antibiotic is selected from the group consisting of Ciprofloxacin, Kanamycin, Oxacillin, Streptomycin, and combinations thereof.

[0232] Illustrative embodiment 39. A food additive, comprising the probiotic composition of any one of Illustrative embodiments 1-21C.

[0233] Illustrative embodiment 40. The food additive of Illustrative embodiment 39, further defined as a food additive for a model organism.

[0234] Illustrative embodiment 41. The food additive of Illustrative embodiment 40, wherein the model organism is C. elegans.

[0235] Illustrative embodiment 42. The food additive of Illustrative embodiment 40, wherein the model organism is selected from the group consisting of rats, mice, rabbits, pigs, hamsters, guinea pigs, dogs, primates, and combinations thereof.

[0236] Illustrative embodiment 43. The food additive of any of Illustrative embodiments 39- 42, further defined as a food additive for a domestic animal.

[0237] Illustrative embodiment 44. A method, comprising the step of administering the food additive of any one of Illustrative embodiments 39-43 to a model organism.

[0238] Illustrative embodiment 45. The method of Illustrative embodiment 44, wherein the food additive extends a life span of the model organism compared to a life span of a model organism that has not received the food additive.

[0239] Illustrative embodiment 46. A method of producing a probiotic composition and / or food additive, comprising the steps of: isolating at least one strain of lactic acid bacterium or a spore thereof from a vegetable source; and formulating the at least one strain of lactic acid bacterium or a spore thereof to provide a probiotic composition and / or food additive.

[0240] Illustrative embodiment 47. The method of Illustrative embodiment 46, wherein the probiotic composition comprises at least one probiotic composition of any of Illustrative embodiments 1-21C.

[0241] Illustrative embodiment 48. The method of Illustrative embodiment 46 or47, wherein the formulating step comprises combining the at least one strain of lactic acid bacterium or spore thereof with at least one pharmaceutically acceptable carrier or excipient.

[0242] Illustrative embodiment 49. The method of Illustrative embodiment 48, wherein the pharmaceutically acceptable carrier or excipient is selected from the group consisting of hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose acetate succinate, microcrystalline cellulose, milk powder, trehalose, xanthan gum, banana powder, dextran,maltose, lactose, glutamic acid, glycerin, PBS, mannitol, partially pregelatinized starch, silicon dioxide, magnesium stearate, maltodextrin, functionalized mineral (Omya), Direct compression grade mannitol, polyvinylpyrrolidone (PVP), dibasic calcium phosphate (DCP), and combinations thereof.

[0243] Illustrative embodiment 50. The method of any of Illustrative embodiments 46-49, further comprising the step of lyophilizing the at least one strain of lactic acid bacterium or a spore thereof.

[0244] Illustrative embodiment 51. The method of any of Illustrative embodiments 46-50, further comprising the step of encapsulating the at least one strain of lactic acid bacterium or a spore thereof.

[0245] Illustrative embodiment 52. The method of Illustrative embodiment 51, wherein the encapsulation method is selected from at least one of the following: microencapsulation (such as for use in chocolate, bakery items, etc.), polymeric encapsulation, spray drying, emulsion, extrusion, spray freeze drying, layer by layer, ionic gelation, vibration technology, sol-gel encapsulation, cyclodextrin inclusion, solvent evaporation method, and combinations thereof.

[0246] Illustrative embodiment 53. The method of any of Illustrative embodiments 46-52, wherein the formulating step comprises combining the at least one strain of lactic acid bacterium or spore thereof at least one prebiotic.

[0247] Illustrative embodiment 54. The method of Illustrative embodiment 53, wherein the at least one prebiotic is selected from the group consisting of [3-glucan, galacto-oligosaccharides, fructo-oligosaccharides, starch, trans-galacto-oligosaccharides, gluco-oligosaccharides, glyco- oligosccharides, lactulose, lactitol, malto-oligosaccharides, xylo-oligosaccharides, stachyose, raffinose, inulin, isomalto-oligosaccharides, xylo-oligosaccharides, lacticol, lactulose, cereal fibre, short-chain oligosaccharide, and combinations thereof.

[0248] Illustrative embodiment 55. The method of any of Illustrative embodiments 46-54, wherein the formulating step comprises producing the probiotic composition in a form selected from the group consisting of a liquid (i.e., drops), a suspension, an emulsion, a dried powder, a capsule, a food product, a medical food, a chewable, a gummy, a spray-dried nasal product, a sachet, or a combination thereof.

[0249] Illustrative embodiment 56. The method of Illustrative embodiment 55, wherein the food product is selected from the group consisting of a yogurt, a kimchi, a dairy product, a fermented food, a yakult drink, a cheese, a chocolate, a bakery item, and combinations thereof.

[0250] Thus, in accordance with the present disclosure, there have been provided devices, kits, and assemblies, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth herein. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

CLAIMS1. A probiotic composition, comprising an effective amount of at least one therapeutic bacterium or a spore thereof, wherein the at least one therapeutic bacterium or spore thereof comprises a strain of lactic acid bacterium or a spore thereof that is isolated from a vegetable source.

2. The probiotic composition of claim 1, wherein the probiotic composition comprises at least two lactic acid bacteria or spores thereof.

3. The probiotic composition of claim 1, wherein the probiotic composition comprises at least one of Lactococcus lactis (L. lactis) and Leuconostoc mesenteroides (L. mesenteroides).

4. The probiotic composition of claim 3, wherein L. mesenteroides has a genomic sequence that is at least about 95% identical to at least one of SEQ ID NOS:l-6, and L. lactis has a genomic sequence that is at least about 95% identical to at least one of SEQ ID NOS:7-12.

5. The probiotic composition of claim 3, wherein the probiotic composition comprises L. lactis and L. mesenteroides.

6. The probiotic composition of claim 5, wherein L. mesenteroides and L. lactis are present in the probiotic composition at a weight ratio in a range of from about 6:1 to about 2:1.

7. The probiotic composition of claim 1, wherein the at least one lactic acid bacterium or spore thereof is isolated from an organic herb.

8. The probiotic composition of claim 1, further comprising at least one pharmaceutically acceptable carrier or excipient.

9. The probiotic composition of claim 1, wherein the strain of lactic acid bacterium or a spore thereof has been lyophilized.

10. The probiotic composition of claim 1, wherein the strain of lactic acid bacterium or a spore thereof is encapsulated.

11. The probiotic composition of claim 1, further comprising at least one prebiotic selected from the group consisting of 0-glucan, galacto-oligosaccharides, fructo-oligosaccharides, starch, trans-galacto-oligosaccharides, gluco-oligosaccharides, glyco-oligosccharides, lactulose, lactitol, malto-oligosaccharides, xylo-oligosaccharides, stachyose, raffinose, inulin, isomaltooligosaccharides, xylo-oligosaccharides, lacticol, lactulose, cereal fibre, short-chain oligosaccharide, and combinations thereof.

12. The probiotic composition of claim 1, wherein the probiotic composition is formulated for oral administration.

13. The probiotic composition of claim 1, wherein the probiotic composition is in a form selected from the group consisting of a liquid (i.e., drops), a suspension, an emulsion, a dried powder, a capsule, a food product, a medical food, a chewable, a gummy, a spray-dried nasal product, a sachet, or a combination thereof.

14. The probiotic composition of claim 1, wherein the at least one therapeutic bacterium or spore thereof of the probiotic composition is substantially resistant to at least one antibiotic.

15. A method, comprising: administering the probiotic composition of any one of claims 1-14 to a subject in need of treatment.

16. The method of claim 15, wherein the subject is a human.

17. The method of claim 15, wherein the subject is a domestic animal.

18. The method of claim 15, wherein the probiotic composition is orally administered to the subject.

19. The method of claim 15, wherein the subject is experiencing at least one gastrointestinal (Gl) symptom selected from the group consisting of upset stomach, heartburn, nausea, vomiting, loose bowel movements, and combinations thereof.

20. The method of claim 15, wherein the subject has an abnormal gut biome.

21. The method of claim 20, wherein administration of at least one antibiotic to the subject has contributed to the abnormal gut biome.

22. The method of claim 15, wherein the subject is immunocompromised.

23. The method of claim 15, wherein the subject has cancer.

24. The method of claim 15, wherein the subject has an autoimmune disease.

25. The method of claim 15, further comprising the step of administering at least one antibiotic to the subject, wherein the at least one therapeutic bacterium or spore thereof of the probiotic composition is at least partially resistant to the at least one antibiotic.

26. A food additive, comprising the probiotic composition of any one of claims 1-14.

27. The food additive of claim 26, further defined as a food additive for a model organism.

28. The food additive of claim 27, wherein the model organism is C. elegans.

29. The food additive of claim 27, wherein the model organism is selected from the group consisting of rats, mice, rabbits, pigs, hamsters, guinea pigs, dogs, primates, and combinations thereof.

30. A method, comprising: administering the food additive of any one of claims 26-29 to a model organism.

31. The method of claim 30, wherein the food additive extends a life span of the model organism compared to a life span of a model organism that has not received the food additive.

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