Probiotic bacteria isolated from phytoplankton biomass, and related compositions and methods

Abstract

Provided is a probiotic bacterium isolated from phytoplankton biomass. The probiotic bacterium can be in the form of at least one isolate. In certain embodiments, the probiotic bacterium is for use as a probiotic in mammals including humans, livestock, and pets. In certain embodiments, the probiotic bacterium can be used to treat or prevent gastrointestinal diseases, gastrointestinal conditions, or gastrointestinal disorders in a subject. Also provided are related compositions comprising the probiotic bacterium and at least one additional component, as well as methods for manufacturing such compositions.

Description

Technical Field

[0001] Related Applications This disclosure claims priority to U.S. Provisional Patent Application No. 63 / 358,336, filed Jul. 5, 2022, the entire content of which is incorporated herein by reference.

[0002] Technical Field This disclosure relates to probiotics. More particularly, this disclosure relates to probiotic bacteria isolated from phytoplankton biomass and related compositions and methods.

Background Art

[0003] Background Most mammalian gastrointestinal (GI) tracts are colonized by indigenous microorganisms that form a microbiota. In healthy mammals, there is a balance between beneficial or benign bacteria and pathogenic bacteria. Intestinal dysbiosis refers to a state in which the balance of the intestinal microbiota is not maintained. Intestinal dysbiosis can cause changes in the balance of immunostimulatory cytokines within the gastrointestinal tract, increased intestinal permeability (leaky gut), proliferation of pathogenic bacteria, increased intestinal transit time that negatively affects nutrient digestion and absorption, effects on the central nervous system, and other systemic health conditions. These changes in immunostimulatory cytokines can, as a result, alter the balance of inflammatory signals within the intestine and cause enteritis as well as other acute and chronic gastrointestinal disorders.

[0004] Probiotics are intended to restore the balance of beneficial bacteria within the gastrointestinal tract in order to treat or prevent intestinal dysbiosis and gastrointestinal disorders. However, while there are numerous commercially available probiotic formulations, not all commercially available probiotics provide beneficial health effects.

Summary of the Invention

[0005] In one aspect, a probiotic bacterium isolated from phytoplankton biomass is provided.

[0006] In certain embodiments, the probiotic bacterium is of the family Lactobacillaceae or Enterococcaceae.

[0007] In certain embodiments, the probiotic bacterium is of the genus Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Leuconostoc, Pediococcus, Lactococcus, Weissella, Enterococcus, Tetragenococcus, or Vagococcus.

[0008] In certain embodiments, the probiotic bacterium is of a species selected from Limosilactobacillus fermentum, Lacticaseibacillus casei, Levilactobacillus brevis, Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus thailandicus, Enterococcus avium, and Pediococcus acidilactici.

[0009] In certain embodiments, the probiotic bacterium is of a species selected from Enterococcus thailandicus, Enterococcus avium, and Limosilactobacillus fermentum.

[0010] In certain embodiments, the probiotic bacteria have a 16S rDNA sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 as determined by sequence alignment performed using BLAST (Basic Local Alignment Search Tool).

[0011] In certain embodiments, the probiotic bacteria have a 16S rDNA sequence that is identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0012] In certain embodiments, the probiotic bacteria are of a strain selected from Limosilactobacillus fermentum FT1-1 (IDAC accession number 050723-01), Limosilactobacillus fermentum FT2-1 (IDAC accession number 050723-02), Lactiplantibacillus casei FT2-3 (IDAC accession number 050723-03), Enterococcus gallinarum FT3-1 (IDAC accession number 050723-04), Limosilactobacillus fermentum FT3-2 (IDAC accession number 050723-05), Enterococcus sp. FT3-4 (IDAC accession number 050723-06), Enterococcus tyndalis FT3-5 (IDAC accession number 050723-07), Enterococcus avium FT3-6 (IDAC accession number 050723-08), Levilactobacillus brevis PN-9 (IDAC accession number 050723-09), or Pediococcus acidilactici PN-10 (IDAC accession number 050723-10).

[0013] In another aspect, there is provided a probiotic bacterium of any of the embodiments disclosed herein for the prevention or treatment of a gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder.

[0014] In certain embodiments, the gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder is selected from intestinal dysbiosis, enteritis, and diarrhea.

[0015] In certain embodiments, the gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder is intestinal dysbiosis.

[0016] In another aspect, there is provided an isolate of a probiotic bacterium of any of the embodiments disclosed herein.

[0017] In another aspect, there is provided a biologically pure culture of a probiotic bacterium of any of the embodiments disclosed herein.

[0018] In another aspect, there is provided a composition comprising a probiotic bacterium of any of the embodiments disclosed herein and at least one additional component.

[0019] In certain embodiments, the at least one additional component comprises prebiotics, additional pharmaceutical or nutritional components, or pharmaceutically or nutritionally acceptable additives.

[0020] In certain embodiments, the composition is in the form of a dietary supplement.

[0021] In certain embodiments, the composition is in the form of a food.

[0022] In another aspect, there is provided a composition of any of the embodiments disclosed herein for the prevention or treatment of a gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder.

[0023] In certain embodiments, the gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder is selected from intestinal dysbiosis, enteritis, and diarrhea.

[0024] In certain embodiments, the gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder is intestinal dysbiosis.

[0025] In another aspect, there is provided a method for manufacturing a composition, the method comprising providing at least one additional component and combining the probiotic bacteria with the at least one additional component.

[0026] In certain embodiments, the at least one additional component is an edible component.

[0027] In certain embodiments, the probiotic bacteria are provided in powder or liquid form.

[0028] In another embodiment, there is provided a kit comprising the probiotic bacteria of any of the embodiments disclosed herein and instructions for use thereof.

[0029] In certain embodiments, the instructions include directions for administering the probiotic bacteria to a subject for treating or preventing a gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder.

[0030] In certain embodiments, the gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder is selected from intestinal dysbiosis, enteritis, and diarrhea.

[0031] In certain embodiments, the gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder is intestinal dysbiosis.

[0032] In certain embodiments, the subject is a mammalian subject.

[0033] In another aspect, there is provided a method for treating or preventing a gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder in a subject, the method comprising administering to the subject a probiotic bacterium of any of the embodiments disclosed herein.

[0034] In certain embodiments, the gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder is selected from intestinal dysbiosis, enteritis, and diarrhea.

[0035] In certain embodiments, the gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder is intestinal dysbiosis.

[0036] In certain embodiments, the subject is a mammalian subject.

[0037] In certain embodiments, the probiotic bacterium is administered orally.

[0038] In another aspect, there is provided the use of a probiotic bacterium of any of the embodiments disclosed herein for treating or preventing a gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder.

[0039] In another aspect, there is provided the use of a probiotic bacterium of any of the embodiments disclosed herein in the manufacture of a medicament for treating a gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder.

[0040] Other aspects and features of the present disclosure will become apparent to those skilled in the art upon review of the following description of specific embodiments of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Some aspects of the present disclosure are described in more detail with reference to the accompanying drawings. In the drawings:

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DETAILED DESCRIPTION OF THE INVENTION

[0042] Detailed description of the embodiments: Generally, the present disclosure provides probiotic bacteria isolated from phytoplankton biomass for use as probiotics. In certain embodiments, the probiotic bacteria are for use as probiotics in mammals including, but not limited to, humans, livestock, and pets. In certain embodiments, the probiotic bacteria can be used to treat or prevent gastrointestinal diseases, conditions, or disorders in a subject. Related compositions, kits, and methods are also provided.

[0043] As used herein, "probiotic bacteria" refers to bacteria that provide at least one beneficial effect to a host organism, and "probiotics" refers to a cell culture or preparation of probiotic bacteria. Beneficial effects on the host organism can include, for example, beneficial effects on the host's digestive system, immune system, and / or brain-gut-microbiota system. Embodiments are not limited by the specific beneficial effects described herein.

[0044] As used herein, "isolated," when used with respect to the probiotic bacteria disclosed herein, refers to bacteria that have been separated from their natural environment.

[0045] As used herein, "phytoplankton biomass" refers to a biological material that includes phytoplankton cells, lysed phytoplankton cells, and / or extracellular material of phytoplankton cells, and may also include other microorganisms such as other types of bacteria. The term "phytoplankton" refers to photosynthetic microorganisms that inhabit aquatic environments and include microalgae and cyanobacteria.

[0046] In certain embodiments, the probiotic bacteria can be isolated from phytoplankton biomass using the methods described in the examples below. In other embodiments, the probiotic bacteria can be isolated from phytoplankton biomass by any other suitable means. For the sake of clarity, it is understood that the probiotic bacteria are not the phytoplankton themselves, but rather other bacteria associated with the phytoplankton biomass.

[0047] In certain embodiments, the probiotic bacteria are in the form of at least one isolate. As used herein, "isolate" or "isolate culture" refers to a culture of probiotic bacteria obtained by culturing a single cell or single colony isolated from its source. In certain embodiments, at least one isolate can be genotypically and / or phenotypically different from other strains of the same species. An isolate can also be referred to as an "archetype strain" because it is an ancient nutrient source for animal life. In certain embodiments, at least one isolate is biologically pure. As used herein, "biologically pure" refers to an isolate that is substantially free of any other biological strain.

[0048] Hereinafter, the probiotic bacteria can be described with respect to isolates and isolate cultures. However, it is understood that the probiotic bacteria of the present disclosure can be in any other suitable form, including as a heterogeneous mixture.

[0049] In certain embodiments, the probiotic bacterium is a lactic acid bacterium (LAB). The probiotic bacterium can be other Lactobacillales. In certain embodiments, the probiotic bacterium is of the species of the genus Lactobacillus, Limosilactobacillus, Lactiplantibacillus, Levilactobacillus, Leuconostoc, Pediococcus, Weissella or any other species of the former genus Lactobacillus (also referred to as "lactobacilli"), including but not limited to those of the species of the family Lactobacillaceae. In certain embodiments, the probiotic bacterium is of the species of the family Enterococcaceae, including, for example, species of the genus Enterococcus, Tetragenococcus or Vagococcus. In other embodiments, the probiotic bacterium is of another species of lactic acid bacteria, including but not limited to species of the genus Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Oenococcus or Sporolactobacillus. In other embodiments, the probiotic bacterium is of any other bacterial genus or species isolated from phytoplankton biomass.

[0050] In certain embodiments, the Rhodotorula species is Rhodotorula fermentum (formerly Lactobacillus fermentum). In certain embodiments, the Lactobacillus casei species is Lactobacillus casei (formerly Lactobacillus casei). In certain embodiments, the Levilactobacillus species is Levilactobacillus brevis (formerly Lactobacillus brevis). In certain embodiments, the Enterococcus species is Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus tyndallii, or Enterococcus avium. In certain embodiments, the Pediococcus species is Pediococcus acidilactici. Those skilled in the art will understand that the current names and the previous names refer to the same species, and the embodiments are not limited to any particular terms.

[0051] In certain embodiments, the probiotic bacteria are selected from the isolates listed in Table 1 below. The Gram staining results showing the bacterial morphology of each isolate in Table 1 are shown in FIGS. 2A - 2C, and the 16S rDNA sequences are shown in FIGS. 6A - 6J.

Table 1

[0052] In certain embodiments, the probiotic bacteria have a 16S rDNA sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 when determined by sequence alignment performed using BLAST (Basic Local Alignment Search Tool). In certain embodiments, the probiotic bacteria have a 16S rDNA sequence that is identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0053] In certain embodiments, the probiotic bacterium is a mutant of one of the strains listed in Table 1. As used herein, "variant" or "mutant strain" refers to a bacterial strain having at least one DNA mutation as compared to the parent (wild-type) strain from which it is derived. DNA mutations can include base substitutions, deletions, insertions, and any other type of natural or induced DNA modification, including transversions and base conversions.

[0054] Biologically pure stocks of each strain in Table 1 were deposited, under the Budapest Treaty, with the International Depository Authority of Canada (IDAC), 1015 Arlington Street, Winnipeg, Manitoba R3E 3R2, Canada, on the dates shown in the table below.

Table 2

[0055] The probiotic bacterium can exhibit probiotic activity in a subject. As used herein, "probiotic activity" refers to the ability of a bacterium to provide at least one beneficial effect in a subject. In certain embodiments, the probiotic bacterium can survive and colonize within the digestive tract of a subject in order to provide a beneficial effect to the subject.

[0056] In certain embodiments, the probiotic bacterium exhibits resistance to pH over a period of time. In certain embodiments, the pH is low, such as when assayed in vitro, indicating that the bacterium may be able to survive passage through the acidic mammalian stomach. As used herein, "low pH" refers to a pH of about 6.9 or less. In certain embodiments, the bacterium is able to survive at a pH of about 2.5 for at least about 6 hours when assayed in vitro. In certain embodiments, the bacterium is able to survive at a pH of about 2.0 for at least about 6 hours when assayed in vitro. As used herein, "survive" means that the viable cell count of the test culture (measured as colony forming units (CFU) per mL) is above the limit of detection, which limit of detection is, for example, [1.7log 10(CFU / mL) or 50 CFU / mL, etc.

[0057] In certain embodiments, the probiotic bacteria are resistant to the presence of bile salts, for example when assayed in vitro, indicating that the bacteria can survive passage through the mammalian intestine. In certain embodiments, the bacteria can survive in the presence of bile salts over a period of time, for example, when assayed in vitro, can survive in the presence of about 3% bile salts for at least about 6 hours.

[0058] In certain embodiments, the probiotic bacteria have the ability to auto-aggregate, for example when assayed in vitro, indicating that the bacterial cells may be able to bind to host intestinal epithelial cells in a subject to facilitate gastrointestinal tract colonization. As used herein, "auto-aggregate" or "auto-aggregating" refers to bacterial cells that bind to each other and aggregate and precipitate out of solution. In certain embodiments, the bacteria have high cell surface hydrophobicity when assayed in vitro, indicating that the bacterial cells may be able to adhere to host cells in a subject to facilitate colonization of the gastrointestinal tract.

[0059] In certain embodiments, the probiotic bacteria have one or more additional properties that support their use as probiotics when assayed in vitro, ex vivo, and / or in vivo. Non-limiting examples of such properties include: the ability to bind to mammalian cells; the ability to modulate the expression of at least one anti-inflammatory cytokine or pro-inflammatory cytokine by host immune cells; the ability to produce one or more inhibitory substances that inhibit the growth of pathogenic and / or spoilage microorganisms; sensitivity to at least one antibiotic; and / or stability over a suitable shelf life.

[0060] In certain embodiments, the probiotic bacteria are in a viable form. In certain embodiments, the probiotic bacteria are in a lyophilized (freeze-dried) form or in the form of a liquid suspension.

[0061] Also provided herein are one or more postbiotics produced by probiotic bacteria. As used herein, "postbiotics" refers to metabolic products or other bioactive compounds produced by microorganisms that confer at least one beneficial effect on a host organism. Postbiotics can be produced intracellularly or extracellularly. In certain embodiments, the probiotic bacteria disclosed herein are provided in combination with one or more postbiotics produced by the probiotic bacteria when grown in a suitable medium. In other embodiments, the probiotic bacteria can be lysed or heat-killed, and the postbiotics can be used in place of the probiotic bacteria in any of the compositions, kits, methods, and / or uses described below.

[0062] Also provided are compositions comprising probiotic bacteria isolated from phytoplankton biomass. Any of the above isolates can be used as the probiotic bacteria in the compositions described herein. In certain embodiments, the composition can comprise two or more isolates of bacteria isolated from phytoplankton biomass. These strains can be derived from the same or different phytoplankton biomass.

[0063] The composition can comprise probiotic bacteria and one or more additional components. In certain embodiments, the composition can further comprise at least one prebiotic. As used herein, "prebiotics" refers to substances that stimulate the growth or activity of at least one beneficial microorganism. In certain embodiments, the prebiotic compound induces the growth or activity of at least one isolate of probiotic bacteria. Non-limiting examples of prebiotics include inulin, pectin, beta-glucan, fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), resistant starch, and organic molecules such as humic and fulvic acids.

[0064] In certain embodiments, the composition may further comprise at least one additional pharmaceutical or nutritional component, such as, for example, at least one vitamin, mineral, fiber, fatty acid, amino acid, or any other suitable pharmaceutical or nutritional component.

[0065] In certain embodiments, the composition may further comprise at least one encapsulating material. Non-limiting examples of encapsulating materials include polysaccharides such as alginic acid, plant / microbial gums, chitosan, starch, k-carrageenan, cellulose acetate phthalate; proteins such as gelatin or milk proteins; and fats. The probiotic bacteria can be encapsulated in the encapsulating material by spray drying, extrusion, gelation, droplet extrusion, emulsion, freeze drying, or any other suitable encapsulation method. Encapsulation of the probiotic bacteria can protect the cells and extend the shelf life of the composition.

[0066] In certain embodiments, the composition is an ingestible composition. As used herein, "ingestible" refers to a substance that can be orally consumed by a subject.

[0067] In certain embodiments, the ingestible composition is in the form of a dietary supplement. The dietary supplement can be in the form of a powder, capsule, gel capsule, microcapsule, bead, tablet, chewable tablet, gummy, liquid, or any other suitable dietary supplement form.

[0068] In certain embodiments, the dietary supplement may further include at least one pharmaceutically or nutritionally acceptable additive. Non-limiting examples of additives include fillers, binders, carriers, diluents, stabilizers, lubricants, glidants, colorants, flavorants, coating agents, disintegrants, preservatives, adsorbents, and sweeteners. In embodiments where the dietary supplement is in the form of a capsule, the dietary supplement may further include a suitable encapsulating material including, but not limited to, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), alginic acid, gelatin, and any other suitable encapsulating agent.

[0069] In certain embodiments, the ingestible composition is in the form of a food. The food can be in any form suitable for mammalian subjects such as human, livestock animals, pets, or any other mammalian subject.

[0070] In certain embodiments, the food is a solid food. The solid food can be in a dry, moist, semi-moist, frozen, dehydrated, freeze-dried, or any other suitable form. In other embodiments, the food is a liquid food. Examples of liquid foods include, but are not limited to, beverages, broths, oil suspensions, gravies, milk-based products, and liquid or semi-solid yogurts.

[0071] In certain embodiments, the ingestible composition is provided in a powder form suitable for sprinkling on the surface of a solid food or mixing into a liquid food. In other embodiments, the ingestible composition is provided in a liquid form suitable for spraying, pouring, or dripping onto the surface of a solid food or mixing with a liquid food. Alternatively, the ingestible composition is in a form suitable for spraying, pouring, or dripping directly into the subject's mouth.

[0072] In other embodiments, the ingestible composition is in the form of a surface coating for a solid food. The surface coating may include a carrier for attaching bacteria to the surface of the solid food. The carrier can include edible oil or any other suitable carrier.

[0073] In another embodiment, the composition can be in a non-ingestible form, such as in the form of a suppository, or any other suitable form.

[0074] Figure 1 is a flowchart of an exemplary method 100 for manufacturing a composition according to some embodiments. At block 102, a probiotic bacterium isolated from a phytoplankton biomass is provided. At block 104, at least one additional component is provided. The term "provide" in this context can refer to manufacturing (including isolating or culturing), receiving, purchasing, or otherwise obtaining the probiotic bacterium and the component. At block 106, the probiotic bacterium is combined with the additional component.

[0075] In certain embodiments, the probiotic bacterium is at least one of the isolates listed in Table 1. In certain embodiments, the probiotic bacterium is in powder form. In other embodiments, the probiotic bacterium is in liquid form.

[0076] In certain embodiments, the component is an edible component. In certain embodiments, the edible component is at least one pharmaceutically or nutritionally acceptable additive as described above for dietary supplements. In other embodiments, the edible component is any of the solid or liquid foods described above or their components.

[0077] In certain embodiments, the probiotic bacteria and the edible ingredient are combined during the manufacture of a nutritional supplement, a solid food, or a liquid food. In other embodiments, the probiotic bacteria are combined with a supplement, a solid food, or a liquid food after manufacture. For example, the probiotic bacteria can be in powder form, and the powder can be sprinkled onto a solid food or mixed with a liquid food or a supplement. As another example, the probiotic bacteria can be in liquid form (e.g., cooking oil), and the liquid can be sprayed, dripped, or coated onto a solid food or mixed with an edible liquid.

[0078] In other embodiments, the probiotic bacteria can be combined with additional ingredients using any other suitable means.

[0079] The probiotic bacteria, isolates, and compositions described herein can be used to improve and / or maintain the health of a subject. The subject can be a mammalian subject including, but not limited to, humans, livestock, and pets. Non-limiting examples of pets include dogs and cats. Non-limiting examples of livestock include cows, sheep, goats, and pigs.

[0080] In certain embodiments, the probiotic bacteria, isolate, or composition can be used to treat or prevent a gastrointestinal disease, disorder, or condition in a subject. As used herein, "treating" or "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a health state or its symptoms, and / or therapeutic in terms of partially or completely curing a health state and / or side effects resulting from the health state. For the sake of clarity, it is intended that the term "treating" or "treatment" in this context includes providing any beneficial physiological effect to a subject, and it is understood that their meaning is not limited to preventing or curing a particular disorder or health condition.

[0081] In certain embodiments, the gastrointestinal disease, disorder or condition includes intestinal dysbiosis. As used herein, "intestinal dysbiosis" refers to any imbalance of the microbiota in the gastrointestinal tract or a part thereof of a subject. In certain embodiments, the gastrointestinal disease, disorder or condition includes enteritis and / or diarrhea. In certain embodiments, the isolate or composition can be used in the manufacture of a medicament for the treatment or prevention of intestinal dysbiosis, enteritis, diarrhea or any other suitable disease, disorder or condition.

[0082] In other embodiments, the probiotic bacteria, isolate or composition can be used to provide any other health benefit to the subject.

[0083] Without being bound by theory, probiotic bacteria isolated from phytoplankton are thought to be similar to ancient food sources for animals. Modern foods (including commercially available pet foods and livestock feeds) have significantly different nutritional profiles compared to ancient food sources, and as a result, changes may have occurred in the gut microbiota. By introducing the "pristine strain" of probiotic bacteria isolated from phytoplankton, the gut microbiota can shift towards a more ancestral state, thereby treating or preventing gastrointestinal conditions such as dysbiosis.

[0084] The probiotic bacteria, isolate or composition can be administered to the subject in an effective amount. As used herein, "effective amount" refers to an amount that provides at least one health benefit to the subject. The effective amount can vary based on numerous factors including, but not limited to, the particular probiotic bacteria (e.g., bacterial strain) being administered and its properties; the age, weight, gender, diet and general health status of the subject; the mode and time of administration; the severity of the condition being treated; the nature of any concurrent treatment or drug therapy; and any other relevant factors.

[0085] In certain embodiments, the probiotic bacteria, isolates or compositions are orally administrable to a subject. In other embodiments, the probiotic bacteria, isolates or compositions may be administered enterally and / or rectally to a subject. The probiotic bacteria, isolates or compositions may be administered to the subject at any suitable interval, including, for example, at least once a month, at least once a week or at least once a day. As an example, about 1 billion CFU per 20 pounds (lbs) of the subject's body weight may be orally administered to the subject once a day.

[0086] Also provided herein is a kit comprising probiotic bacteria isolated from a plant plankton biomass contained in a container and instructions for its use. In certain embodiments, the kit comprises one or more isolates of the probiotic bacteria. In certain embodiments, the kit comprises a composition comprising a probiotic bacteria, such as one or more of the isolates. The isolate(s) and / or composition may be any embodiment of the isolates and compositions disclosed herein.

[0087] The instructions may include, for example, instructions for administering the probiotic bacteria to a mammalian subject for treating or preventing gastrointestinal diseases, disorders or conditions including, but not limited to, intestinal dysbiosis, enteritis and / or diarrhea. The instructions may include recommended dosages and frequencies for administration and may also include instructions such as to ingest the probiotic bacteria with food or without food, with other drug therapies or without other drug therapies. In certain embodiments, the instructions include instructions for combining the probiotic bacteria with a solid or liquid food.

[0088] Without limitation to the foregoing, the bacterial strains, compositions, uses and methods of the present invention are further illustrated by the following examples.

Examples

[0089] Example 1 - Isolation of Bacteria and Extraction of Genomic DNA Adored Beast Apothecary LtdTM Lactic acid bacteria (LAB) were isolated from three bags of freeze-dried phytoplankton products provided by Fitoplankton Marino, El Puerto de Santa Maria, Cadiz, Spain. TM The phytoplankton products were marine microalgae products obtained from Fitoplankton Marino. The powder in each bag was dissolved in 10-fold volume of 0.1% peptone water (PW). Since the weight of the powder was 0.5 g, the volume of 0.1% PW was 4.5 mL. Then, the dilution was vortexed for 1 minute to confirm that the solution was completely homogenized. After homogenization, 100 μL of the dilution was spread on De Man, Rogosa, Sharpe (MRS) agar medium (Oxoid Limited TM , Canada) and then cultured at 37 °C for 24 - 48 hours under aerobic conditions. To obtain a sufficient amount of bacteria for downstream tests, first, individual colonies with different morphologies were picked and inoculated on a new MRS agar medium plate at 37 °C for 24 - 48 hours under aerobic conditions. Then, the bacterial strains that were successfully isolated were transferred to MRS broth and cultured overnight at 37 °C for 24 hours. The isolates were stored at -80 °C in MRS broth + 20% glycerol (El-Soda et al., 2003).

[0090] To extract genomic DNA (gDNA) from all isolates, candidate strains were cultured overnight at 37 °C in MRS broth. Then, 5 mL of the overnight culture was collected at 8,000 rpm for 5 minutes and extracted. The protocol was as follows: The cell pellet was resuspended and washed twice with 500 μL of T100E, and then 10 μL of lysozyme stock solution (10 mg of lysozyme dissolved in 0.5 mL of ddH2O) was added. The lysate was incubated at 37 °C for more than 3 hours, and at the end of incubation, centrifuged at 10,000 rpm for 5 minutes. Then, the cell pellet was homogenized with 500 μL of T100E buffer. 50 μL of 20% SDS (sodium dodecyl sulfate) was added and the tube was inverted 5 - 7 times. 300 μL of sodium acetate solution (3 M, pH 5.2) was added to the lysate and immediately inverted 5 - 7 times. Then, the lysate was incubated at 4 °C for 20 minutes and pelleted at 13,000 rpm for 10 minutes. The supernatant was collected and mixed with an equal volume of ice-cold isopropanol. The gDNA was precipitated by centrifuging at 13,000 rpm for 20 minutes and washed with 600 μL of 70% ethanol. The recovered gDNA product was air-dried for 15 minutes and rehydrated with 50 μL of nuclease-free H2O.

[0091] Example 2 - Identification of Isolates Gram Staining of Isolates Gram staining was performed using a BD BBL TM Gram staining kit (Cat#: B4312539, Fisher Scientific TM , Canada), and images were taken with ScopeImage TM and a microscope. The results of Gram staining are shown in FIGS. 2A - 2C.

[0092] Based on the typical morphological appearance, a total of 35 LAB strains were isolated from three bags of phytoplankton products. All 35 strains were Gram-positive bacteria. The bacterial strains were assigned unique strain IDs according to the bags from which they were isolated. For example, the strain ID "1-2" means the second strain isolated from bag #1. The second isolation was carried out from bag #1, and the bacterial strains were assigned unique strain IDs according to the source from which each strain was isolated. For example, the strain ID PN-9 means the 9th strain isolated from phytoplankton (PN).

[0093] Molecular Identification of Isolates The molecular identification of LAB strains was first carried out using M13 primers (Rossetti, L, 2005), and its sequence was 5'-GAG GGT GGC GGT TCT -3' (Ta(℃)=45℃). To obtain the fingerprint profile of the isolates, the amplification reaction was carried out using RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) technology in a total volume of 25 μL with a program of 94℃ for 5 minutes; 40 cycles of 94℃ for 1 minute, 45℃ for 1 minute, and 72℃ for 2 minutes; 72℃ for 5 minutes and stored at 4℃. Figures 3A and 3B show the RAPD profiles of 33 out of 35 isolated LAB strains.

[0094] To identify the isolates at the species level, the gene encoding 16S ribosomal RNA (rRNA) was amplified by PCR and sequenced by Sanger sequencing. The DNA sequence encoding 16S rRNA is called the 16S rDNA sequence.

[0095] To amplify the conserved regions of the 16S rRNA gene, a PCR reaction was performed using 16S rDNA primers. The primer sequences were: 16S-8F: 5'-AGA GTT TGA TCC TGG CTC AG-3'; 16S-805R: 5'-GAC TAC CAG GGT ATC TAA TCC-3' (Ta(℃)=55℃). The PCR was programmed as follows: 5 minutes at 95℃; 35 cycles of 30 seconds at 95℃, 30 seconds at 55℃, and 30 seconds at 72℃; 10 minutes at 72℃ and stored at 4℃. The product obtained from the 16S rDNA PCR reaction was purified using the GeneJET TM PCR Purification Kit (Cat#: K0701, Thermo Scientific TM , Canada) and submitted to the Molecular Biology Services Unit (MBSU) (Department of Biological Sciences, University of Alberta) for sequencing. Finally, to identify the LAB strains, the sequencing data was further aligned in the Genbank TM database using the basic local alignment search tool (BLAST, www.ncbi.nlm.nih.gov / BLAST). The reagents and equipment used in this study were as follows: The Taq polymerase used in this study was DreamTaq TM DNA Polymerase (Cat#: EP0703, Thermo Scientific TM , Canada); The PCR products were analyzed by electrophoresis and imaged and photographed by transmission illumination under ultraviolet light using an AlpHaImager TM gel imaging device (ThermoFisher, Canada).

[0096] Figures 4A and 4B show the agarose gel electrophoresis of 16S rDNA PCR products for 33 out of 35 isolated LAB strains.

[0097] The putative identification of the species of each strain based on the BLAST alignment is shown in Tables 3, 4, and 5 below.

Table 3

Table 4

Table 5

[0098] Twenty-two strains were Lactobacillus fermentum, two strains were Lactobacillus casei subsp. casei, one strain was Levilactobacillus brevis, one strain was Pediococcus acidilactici, and nine strains were Enterococcus. Most of the Enterococcus were isolated from bag #3, while most of the Lactobacillus casei subsp. casei and Lactobacillus fermentum were isolated from bags #1 and #2. As shown in Figure 5, using M13 primers, RAPD profiling of one representative strain of Lactobacillus casei subsp. casei and Lactobacillus fermentum from each bag was performed again, with K9-1 (Lactobacillus casei subsp. casei) and K9-2 (Lactobacillus fermentum) as positive controls.

[0099] The 16S rDNA sequencing results for strains FT1-1 (Lactobacillus fermentum), FT2-1 (Lactobacillus fermentum), FT2-3 (Lactobacillus casei subsp. casei), FT3-1 (Enterococcus gallinarum), FT3-2 (Lactobacillus fermentum), FT3-4 (Enterococcus), FT3-5 (Enterococcus tyndalensis), FT3-6 (Enterococcus avium), PN-9 (Levilactobacillus brevis), and PN-10 (Pediococcus acidilactici) are shown in Figures 6A - 6J. These strains were deposited on July 5, 2023, with the International Depository Authority of Canada (IDAC), 1015 Arlington Street, Winnipeg, Manitoba R3E 3R2, Canada, under the Budapest Treaty and were assigned the accession numbers shown in Table 2 above.

[0100] Example 3 - Characterization of Isolates The biological activities of eight strains (FT1-1, FT2-1, FT2-3, FT3-1, FT3-2, FT3-4, FT3-5 and FT3-6) were characterized using the method described in International PCT Application No. PCT / CA2019 / 051140, which is hereby incorporated by reference.

[0101] Autoaggregation Ability To evaluate the autoagglutination activity of the isolates, an autoagglutination assay was performed. A sample of the fully grown culture was thoroughly mixed by vortexing. The initial optical density at 600 nm (OD 600 , A0) was measured and recorded. The remaining cell suspension was left standing at ambient temperature for 4 hours. An aliquot of the upper suspension (the cell suspension was not vortexed) was taken and the OD 600nm (A t ) was measured. The autoagglutination rate is expressed as follows:

Equation

Table 6

[0102] Cell Surface Hydrophobicity To evaluate the hydrophobicity of the bacterial cell surface of each isolate, a microbial adhesion to hydrocarbons (MATH) assay (Otero et al., 2004) was performed to measure the hydrophobicity of the bacterial strains from the perspective of adhesion. The fully grown culture samples were collected by centrifugation and then the cells were washed with physiological saline. The cell pellet was resuspended in physiological saline and the OD 600 of each cell suspension was adjusted. The actual final OD 600It was measured and recorded. An aliquot of the cell suspension was combined with a solvent (toluene or xylene) in a test tube and vortexed vigorously for 1 minute. The test tube was allowed to stand for 1 hour to separate the immiscible solvent and the aqueous layer. The aqueous layer was removed, and OD 600 (OD 試験 ) was measured and recorded. The hydrophobicity rate of each strain was calculated as follows:

Equation

[0103] The hydrophobicity rates of the 7 isolates are shown in Table 7 below. These results indicate that the isolates may have the potential to adhere to the surface of host intestinal epithelial cells.

Table 7

[0104] Low pH Tolerance Assay To evaluate the resistance of the isolates to acidic conditions, for each isolate, an aliquot of the fully grown culture was subcultured in simulated gastric fluid (SGF, without pepsin) at pH = 2.0 and pH = 2.5. SGF solutions with various pH values were prepared by adjusting the pH of SGF with HCl and NaOH, and then sterilized by filtration. After inoculating the cultures into each SGF solution, the mixture was thoroughly mixed by vortexing, and an aliquot was taken at 0 hours for dilution and plating. The remaining culture was immediately cultured at 37°C for 6 hours under sealed conditions, and then an aliquot was taken for dilution and plating.

[0105] Serial 10-fold dilutions of each culture were prepared, and the dilutions were plated on MRS agar plates and cultured at 37°C for 2 days. The number of viable cells was recorded, and the average of at least 3 independent replicates [log 10(CFU / mL)] was expressed as the mean ± standard deviation. The results of the low pH tolerance assay for the 8 isolates are shown in Table 8 below, where 1 = the strain survived at pH 2 and pH 2.5 after 6 hours; 2 = the strain did not survive at pH 2 but survived at pH 2.5 after 6 hours; 3 = the strain did not survive at pH 2 and pH 2.5 after 6 hours.

Table 8

[0106] The results show that all strains can survive for 6 hours at pH 2.5, and strains FT3-2, FT3-5, and FT3-6 can also survive for 6 hours at pH 2.0.

[0107] Bile Salt Tolerance Assay To evaluate the tolerance of the isolates to bile salts, for each isolate, an aliquot of the fully grown culture was subcultured into phosphate-buffered saline (PBS, pH = 7.2) solutions with varying bile salt concentrations (0%, 3%, and 5%). PBS solutions with various bile salt concentrations were prepared by dissolving the corresponding amounts of bile salts in sterile PBS. After inoculating the cultures into each PBS solution, the mixture was thoroughly mixed by vortexing, and 0-hour aliquots were taken for dilution and plating. The remaining culture broth was immediately cultured at 37°C under sealed conditions for 24 hours. Aliquots were taken at 6 hours and 24 hours for dilution and plating.

[0108] Each culture broth was serially diluted 10-fold, and appropriate dilutions were plated onto MRS agar plates and cultured at 37°C for 2 days. The number of viable cells was recorded, and the mean [log of at least 3 independent replicates was calculated. 10(CFU / mL)] was expressed as ± standard deviation. The results of the bile salt tolerance assay for 8 isolates are shown in Table 9 below, where 1 = the strain survived at 0 hours, 6 hours, and 24 hours without bile; 2 = the strain survived at 0 hours in 3% bile; 3 = the strain survived at 6 hours in 3% bile; 4 = the strain survived at 24 hours in 3% bile; 5 = the strain survived at 0 hours in 5% bile; 6 = the strain survived at 6 hours in 5% bile; 7 = the strain survived at 24 hours in 5% bile. [Table 9]

[0109] The results indicate that strains FT3-5 and FT3-6 can survive in 3% bile for 6 hours.

[0110] Although specific embodiments are shown and described, it will be understood by those skilled in the art that various changes and modifications can be made without departing from the scope of the present disclosure. The terms and expressions used in the foregoing specification are used herein as terms of description rather than limitation, and in the use of such terms and expressions, there is no intention to exclude equivalents of the features shown and described or portions thereof. Further, in the interpretation of the present disclosure, all terms should be construed as broadly as possible in accordance with the context. In particular, the term "comprising" should be construed as referring to elements, components, or steps in a non-exclusive manner, indicating that the recited elements, components, or steps may exist, be utilized, or be combined with other elements, components, or steps not explicitly recited.

[0111] References The following references are hereby incorporated by reference in their entirety: El Soda, M., Ahmed, N., Omran, N., Osman, G., & Morsi, A. (2003). Isolation, identification and selection of lactic acid bacteria cultures for cheesemaking. Emirates Journal of Food and Agriculture, 51-71; Rossetti, L., & Giraffa, G. (2005). Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases. Journal of microbiological methods, 63(2), 135-144; Otero et al. (2004) “Bacterial surface characteristics applied to selection of probiotic microorganisms”, in Public Health Microbiology, pp. 435-440. Humana Press。

Claims

1. Probiotic bacteria isolated from phytoplankton biomass.

2. The probiotic bacteria according to claim 1, wherein the probiotic bacteria include lactic acid bacteria.

3. The probiotic bacterium according to claim 2, wherein the probiotic bacterium belongs to the Lactobacillaceae family or the Enterococcusae family.

4. The probiotic bacteria according to claim 3, wherein the probiotic bacteria belong to the genera Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Levilactobacillus, Leuconostoc, Pediococcus, Lactococcus, Weissella, Enterococcus, Tetragenococcus, or Vagococcus.

5. The probiotic bacterium according to claim 4, wherein the probiotic bacterium is a species selected from Limosilactobacillus fermentum, Lacticaseibacillus casei, Levilactobacillus brevis, Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus thailandicus, Enterococcus avium, and Pediococcus acidilactici.

6. The probiotic bacterium according to claim 5, wherein the probiotic bacterium is a species selected from Enterococcus tylandis, Enterococcus avium, and Rimosilactobacillus fermentum.

7. The probiotic bacterium according to claim 1, having a 16S rDNA sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 when determined by sequence alignment performed using BLAST (Basic Local Alignment Search Tool).

8. The probiotic bacterium according to claim 7, wherein the probiotic bacterium has the same 16S rDNA sequence as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:

10.

9. The probiotic bacteria include Rimosilactobacillus fermentum FT1-1 (IDAC accession number 050723-01), Rimosilactobacillus fermentum FT2-1 (IDAC accession number 050723-02), Lacticaseibacillus casei FT2-3 (IDAC accession number 050723-03), Enterococcus gallinarum FT3-1 (IDAC accession number 050723-04), Rimosilactobacillus fermentum FT3-2 (IDAC accession number 050723-05), Enterococcus sp. FT3-4 (IDAC accession number 050723-06), and Enterococcus tylandis. The probiotic bacterium according to claim 1, wherein the strain is selected from FT3-5 (IDAC accession number 050723-07), Enterococcus avium FT3-6 (IDAC accession number 050723-08), Reviractobacillus brevis PN-9 (IDAC accession number 050723-09), or Pediococcus acidilactici PN-10 (IDAC accession number 050723-10).

10. An isolated strain of probiotic bacteria according to any one of claims 1 to 9.

11. A biologically pure culture of the isolated strain according to claim 10.

12. A composition comprising the probiotic bacteria described in any one of claims 1 to 9 and at least one further component.

13. The composition according to claim 12, wherein at least one further component comprises a prebiotic, a further pharmaceutically or nutritionally acceptable ingredient, or a pharmaceutically or nutritionally acceptable additive.

14. The composition according to claim 12, wherein the composition is in the form of a nutritional supplement.

15. The composition according to claim 12, wherein the composition is in the form of a food product.

16. The composition according to claim 12 for the prevention or treatment of digestive diseases, digestive conditions, or digestive disorders.

17. The composition according to claim 16, wherein the digestive disease, digestive condition, or digestive disorder is selected from intestinal dysbiosis, enteritis, and diarrhea.

18. The composition according to claim 17, wherein the digestive disease, digestive condition, or digestive disorder is intestinal dysbiosis.

19. A method for producing a composition, To prepare the probiotic bacteria according to any one of claims 1 to 9; To prepare at least one additional component; and Combining probiotics with at least one additional ingredient The method, including the method.

20. The method according to claim 19, wherein at least one further component is an edible component.

21. The method according to claim 19, wherein the probiotic bacteria are provided in powder or liquid form.

22. A kit comprising the probiotic bacteria described in any one of claims 1 to 9 and instructions for use thereof.

23. The kit according to claim 22, wherein the instructions include instructions for administering probiotic bacteria to a subject to treat or prevent a gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder.

24. The kit according to claim 23, wherein the gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder is selected from intestinal dysbiosis, enteritis, and diarrhea.

25. The kit according to claim 24, wherein the digestive disease, digestive condition, or digestive disorder is intestinal dysbiosis.

26. The kit according to claim 23, wherein the target is mammals.

27. A pharmaceutical agent for treating or preventing a gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder in a subject, comprising the probiotic bacteria described in any one of claims 1 to 9.

28. The pharmaceutical product according to claim 27, wherein the digestive disorder, digestive condition, or digestive disorder is selected from intestinal dysbiosis, enteritis, and diarrhea.

29. The pharmaceutical product according to claim 28, wherein the digestive disease, digestive condition, or digestive disorder is intestinal dysbiosis.

30. The pharmaceutical product according to claim 27, wherein the target is a mammal.

31. The pharmaceutical product according to claim 27, which is administered orally.

32. Use of probiotic bacteria according to any one of claims 1 to 9 in the manufacture of a pharmaceutical product for the treatment of a gastrointestinal disease, gastrointestinal condition, or gastrointestinal disorder.

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