Liquid condensates for targeted delivery or removal of microbial cells and molecules

Abstract

The present invention relates to polypeptides, liquid peptide condensates and compositions thereof acting as an agent to provide substance to support or improve overall health such as for the prophylactic or therapeutic treatment of mucosal disorders, epithelial disorders, and / or gut disorders. In one embodiment, a polypeptide is derived from surface layer protein of Lactobacillus acidophilus cells, and use of the polypeptide to aggregate, associate and encapsulate microbial cells for e.g. delivering probiotic microorganism or separating pathogenic microorganism.

Description

[0001] Liquid Condensates for Targeted Delivery or Removal of Microbial cells and Molecules

[0002] Technical field

[0003] The present invention relates, in general terms, to microbiology. In particular, the specification describes polypeptides, liquid condensates and compositions thereof acting as an agent to provide substance to support or improve overall health such as for the prophylactic or therapeutic treatment of mucosal disorders, epithelial disorders, and / or gut disorders.

[0004] Background

[0005] The human gastrointestinal (GI) system hosts a vast reservoir of microbes, totalling approximately 1014functional bacterial cells. These include pathogenic bacteria such as Clostridium difficile and Streptococcus spp. that can lead to diseases. Live microbial cells, delivered in adequate amounts to exert a beneficial effect(s) on the host, are referred to as probiotics.

[0006] When administered, these beneficial bacteria have the potential to establish themselves and foster a healthy microbial ecosystem with the gut being the system most extensively explored for probiotic usage. The administration of probiotics helps replenish what is naturally depleted due to diseases and environmental factors including diet, medications, lifestyle choices, extreme pH levels, digestive enzymes in GI fluids, and bile salts. Moreover, the proximity of beneficial bacteria to the target site has been shown to influence the suppression and elimination of harmful pathogens.

[0007] Presently, methods for delivering probiotic bacteria, mainly Lactobacillus spp. and Bifidobacterium spp., lack specificity and typically require a high microbial load to exert their effects. The sensitivity of these microbes to variations in pH and exposure to digestive enzymes during their journey through the GI tract poses an additional significant challenge. A common approach to oral probiotics delivery involves encapsulating them in gastro- resistant polymers, like alginate or its derivatives, to protect the microbes from environmental threats post-administration. Additionally, certain food products, such as cheese, milk, and yogurt, serve as suitable carriers for probiotic bacteria. Nevertheless, the lack of specificity in microbial colonization and low cell retention in the gut greatly hampers the effectiveness of these formulations.

[0008] It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

[0009] Summary

[0010] Disclosed herein is a polypeptide comprising or consisting of an amino acid sequence having at least 70% (including 80%, 90%, 95% or 100%) sequence identity to SEQ ID NO: 1, or a fragment thereof, wherein the polypeptide or fragment thereof is capable of forming a liquid peptide condensate.

[0011] Disclosed herein is a liquid peptide condensate, comprising a polypeptide as defined herein.

[0012] Disclosed herein is a composition comprising i) a polypeptide or a liquid peptide condensate as defined herein, and ii) a microorganism.

[0013] Disclosed herein is a composition comprising i) a polypeptide or a liquid peptide condensate as defined herein and a suitable functional biological or chemical agent, wherein the agent is selected from a group comprising a drug, a vaccine, a small molecule, a prebiotic, a nanoparticle, a bacteriocin, an antibody, and a combination thereof.

[0014] Disclosed herein is the use of a polypeptide or the liquid peptide condensate as defined herein for aggregating probiotic microorganism for probiotic delivery.

[0015] Disclosed herein is the use of a polypeptide or the liquid peptide condensate as defined herein for aggregating pathogenic microorgani m.

[0016] Disclosed herein is a composition as defined herein for use in treating and / or preventing a mucosal, epithelial and / or gut condition or disease in a subject. Disclosed herein is the use of a composition as defined herein in the manufacture of a medicament for treating and / or preventing a mucosal, epithelial, and / or gut condition or disease in a subject.

[0017] Disclosed herein is a method of treating and / or preventing a mucosal, epithelial, and / or gut condition or disease in a subject, the method comprising administering a composition as defined herein to the subject.

[0018] Disclosed herein is a method of producing a liquid peptide condensate, wherein the method comprises (i) isolating the polypeptide as defined herein; (ii) contacting the polypeptide with a suitable aqueous solution; and (iii) adjusting the pH to suitable range.

[0019] Brief description of the drawings

[0020] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0021] Figure 1. Short peptide sequence (REP6) of L. acidophilus PCB 001 S-layer protein undergoes phase separation into liquid condensates. (A) Line chart in the upper panel demonstrated identification of seven regions with higher likelihood of being intrinsically disordered across the entire L. acidophilus PCB 001 S-layer protein amino acid sequence. The lower panel represents several recombinantly expressed protein (REP) domains of S- layer protein across the full-length protein as represented by the bar chart. The left-hand axis of the bar chart summarizes the outcomes of the liquid-liquid phase separation (LLPS) screening assay, with ‘Y’ (Yes) and ‘N’ (No) indicating whether liquid condensates formed following preincubation of REP in buffer. Bottom panel demonstrates REP6 liquid condensates formed at different salt and peptide concentrations. (B) Images showing no droplet formation for REP1, REP2, REP4 and REP5 under similar conditions. (C) (Left) Images showing green fluorescence protein (GFP)-tagged REP6 forms liquid condensates, and (Right) the liquid condensates layer on the artificial cell surfaces.

[0022] Figure 2. Strong association of REP6 liquid condensates with different bacterial. A, B, E, F and G show single strain Lactobacillus bacteria association and aggregation with REP6 liquid condensates. H shows simultaneous association of peptide droplets with two strains of Lactobacillus bacteria. C and D are the negative controls of A in the absence of REP6 liquid condensates in the solution. I and J show lack of association of two non-lactic acid bacteria (rod shape, marked with white arrows) with peptide liquid condensates. K-0 demonstrates REP6 liquid condensates mediated aggregation and / or encapsulation of pathogenic cells.

[0023] Figure 3. (A) P. aeruginosa cells (light grey in rod shape) show a pH-dependent association with REP6 liquid condensates at acidic pH (i.e., pH<6). (B-C) The REP6 liquid droplets wetted lipid membranes. White arrows mark the REP6-GFP on the lipid membranes. (D) Lactic acid bacteria induced REP6 liquid condensation and cellular aggregation (E) Nanoparticles (e.g., 50nm nanoplastics, grey with higher luminance) exhibit greater aggregation to the surface of peptide liquid condensates. (F) REP6 liquid condensates encapsulating fluorophore-conjugated antibody (light grey).

[0024] Figure 4, Viability of Lactobacillus cells: (A) in buffer alone or with REP6 added, showing no significant difference in the viability after overnight storage at 4°C; in simulated (B) gastric juice (pH 2) and (C) intestinal fluid (pH 7) compared to the corresponding buffer control. Statistical analysis of the viable cell count was performed using Student’s t test. (D- E) Preformed Lactobacillus ccll-REP6 liquid condensates remained as aggregates (D) 3min and (E) 60 min after resuspension in simulated gastric juice (pH 2 with pepsin).

[0025] Figure 5, REP6 liquid condensates mediate adhesion of bacterial cell aggregates to the mucin layer. (A) In the absence of REP6 liquid condensates, L. acidophilus PCB 001 adhered to mucin layer as individual cells or in pairs (black arrows). (B) L. acidophilus PCB 001 (rod shape, white arrows) adhered onto mucin layer as cellular aggregates in the presence of REP6 liquid condensates (grey with higher luminance, sphere).

[0026] Detailed description

[0027] The present disclosure discloses a Lactobacillus cell-derived peptide (REP6), the peptide comprising 276 amino acid residues from full-length S-layer protein of L. acidophilus ATCC4356 or PCB 001.

[0028] Disclosed herein is a polypeptide comprising or consisting of an amino acid sequence having at least 70% (including 80%, 85%, 90%, 95% or 100%) sequence identity to:

[0029] ASYNGKTYTANLKADTENATITAAGSTTAVKPAELAAGVAYTVTVNDVSFNFGS ENAGKTVTLGSANSNVKFTGTNSDNQTETNVSTLKVKLDQNGVASLTNVSIANV YAINTTDNSNVNFYDVTSGATVTNGAVSVNADNQGQVNVANVVAAINSKYFAA QYADKKLNTRTANTEDAIKAALKDQKIDVNSVGYFKAPHTFTVNVKATSNTNGK SATLPVVVTVPNVAEPTVASVSKRIMHNAYYYDKDAKRVGTDSVKRYNSVSVLP NETTING (SEQ ID NO: 1), or a fragment thereof, wherein the polypeptide or fragment thereof is capable of forming a liquid peptide condensate.

[0030] The polypeptide or fragment thereof is capable of spontaneously forming a liquid peptide condensate under suitable conditions.

[0031] Provided herein is a liquid peptide condensate, comprising a polypeptide as defined herein.

[0032] Without being bound by theory, the inventors have demonstrated the use of a Lactobacillus cell-derived peptide to aggregate, associate and encapsulate bacteria. This can be used for the delivery of microbiome-modulating agents. This preparation can deliver bacteria in the form of microcolonies, while providing polymer material (i.e., the Lactobacillus cell-derived peptide) capable of facilitating cellular aggregation, thereby promoting biofilm formation and enhancing cellular colonization. As a result, this approach can increase the adhesion of cells particularly to those non-aggregating and / or non-adhesive cells for successful colonization of the intended site. Additionally, the association of the cell-derived peptide liquid condensates to various drugs, chemical and biological compounds can expand the application to the delivery of these compounds to the targeted site of interest or for the isolation and separation of molecules or cells of interest. The peptide is naturally derived and found within the host and hence not foreign to the body. Elcrc, these peptide liquid condensates can be seen as a natural alternative to currently utilized chemical compounds in the similar fields of application.

[0033] In one embodiment, the polypeptide or fragment thereof does not comprise an amino acid sequence having at least 70% (including 80%, 90%, 95% or 100%) sequence identity to MKKNLRIVSAAAAALLAVAPVAASAVSTVSAATTINASSSAINTNTNAKYDVDVT PSVSAVAANTANNTPAIAGNLTGTIS (SEQ ID NO: 9). In one embodiment, the polypeptide or fragment thereof comprises or consists of an amino acid sequence having at least 70% (including 80%, 85%, 90%, 95% or 100%) sequence identity to SEQ ID NO: 1, or a fragment thereof, wherein the polypeptide is capable of forming a liquid condensate.

[0034] In one embodiment, the polypeptide or fragment thereof comprises or consists of an amino acid sequence having at least 70% sequence identity to an amino acid sequence starting from position 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,

[0035] 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,

[0036] 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 or 132 and ending with position 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,

[0037] 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,

[0038] 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356 or 357 of:

[0039] MKKNLRIVSAAAAALLAVAPVAASAVSTVSAATTINASSSAINTNTNAKYDVDVT PSVSAVAANTANNTPAIAGNLTGTISASYNGKTYTANLKADTENATITAAGSTTAV KPAELAAGVAYTVTVNDVSFNFGSENAGKTVTLGSANSNVKFTGTNSDNQTETN VSTLKVKLDQNGVASLTNVSIANVYAINTTDNSNVNFYDVTSGATVTNGAVSVN ADNQGQVNVANVVAAINSKYFAAQYADKKLNTRTANTEDAIKAALKDQKIDVN SVGYFKAPHTFTVNVKATSNTNGKSATLPVVVTVPNVAEPTVASVSKRIMHNAY YYDKDAKRVGTDSVICRYNSVSVLPNTTTING (SEQ ID NO: 8)

[0040] The term “identity” of sequence as used herein is interchangeably used with “homology”, and refers to a similarity degree between sequences as measured by sequence alignment softwares, such as BLAST. The sequence alignment methods and softwares are well-known by those skilled in the art. Modified amino acid sequences can be obtained by substitution, deletion and / or addition of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more) amino acids or bases in a known sequence. For example, by modifying the amino acid sequence as set forth in the present disclosure via conventional means (for example, by conservative substitution), it is feasible to obtain sequences having more than 70%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% sequence identity to these sequences, and having substantially the same properties, which arc encompassed within the protective scope of the present disclosure. In some embodiments, the present invention contemplates variants of the polypeptide molecule of the liquid condensate of the present invention, wherein the variant has a considerable degree of identity, e.g., identity of at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher, relative to the polypeptide specifically disclosed herein and the sequences thereof. The variant may comprise conservative modifications.

[0041] In some embodiments, the REP6 polypeptide comprises a tag for purification, such as a His- tag or a glutathione S-transferase. The tag for purification can be on the N-terminal of the REP6 polypeptide or the C-terminal of the REP6 polypeptide, or internal to the REP6 polypeptide. In some embodiments, the REP6 polypeptide comprises a tag for visualization, such as fluorescent protein. Non-limiting examples of fluorescent proteins include green fluorescent protein (GFP), enhanced GFP (eGFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), tdTomato, mChcrry, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and infrared fluorescent protein (iRFP). The tag for visualization can be on the N-terminal of the REP6 polypeptide or the C-terminal of the REP6 polypeptide, or internal to the REP6 polypeptide.

[0042] The term “target” refers to prime objective of pharmacology, such as cytotoxic drugs targeting tumor cells, immunomodulatory drugs targeting regulators of the immune system, microbial colonics targeting tissues or organs or a part thereof, and chemical ablation agents targeting tissues. The terms “target region”, “target sites” and “targeted sites” refers to the spatial area where the drug is administered to the target, such as the organ or a portion thereof. Non-limiting examples of target sites include gastrointestinal epithelium mucus layer, mucosal layer of the gut lining, mucosal layer of the oral cavity and mucosal layer of the anus.

[0043] In some embodiments, the polypeptide as defined herein forms a liquid condensate that serve as a medium for aggregating, encapsulating, adhering and / or separating microorganisms. The aggregation process within the peptide liquid droplets can be controlled by various environmental parameters (including but not limited to the pH of the liquid medium, the charge of the liquid condensate surface, and the presence of different types of microorganisms such as probiotic and pathogenic bacteria).

[0044] The microbial cells can be selectively associated on the periphery of the S-laycr derived peptide liquid droplets / condensates. The S-layer derived peptide liquid droplets may determine which microorganisms are selectively encapsulated and the degree of encapsulation can be controlled through environmental factors such as pH.

[0045] In one embodiment, the S-layer derived liquid condensate is attracted to biological surfaces and facilitates the delivery of aggregated, associated and encapsulated entities (i.e., microorganisms or bioactive compounds) to biological surfaces including but not limited to epithelial cells.

[0046] The peptide liquid condensates may have no significant impact on the viability of the aggregated, associated and encapsulated microbes. hi one embodiment, the liquid condensates serve as carriers and / or encapsulating agents for the delivery and / or protection of drugs, chemical and biological molecules, such as, antibodies, vaccines, liposomes and antimicrobial agents.

[0047] In one embodiment, the liquid condensates serve as an aggregating agent of small particles including nanoparticles.

[0048] The peptide liquid condensates can be utilized for separation, fractionation, concentration and purification of molecules, particles, cells and microbes.

[0049] The terms "protein" and "polypeptide" are used interchangeably and refer to any polymer of amino acids (dipeptide or greater) linked through peptide bonds or modified peptide bonds. Polypeptides of less than about 10-20 amino acid residues are commonly referred to as "peptides." Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogues of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages. The polypeptides of the invention may comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a polypeptide by the cell in which the polypeptide is produced, and will vary with the type of cell. Polypeptides are defined herein, in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

[0050] The term “amino acid sequence” refers to an arrangement in which amino acids are linked to each other to form a peptide chain (or polypeptide), wherein the amino acid sequence can only be read in one direction. There are more than 100 types of different amino acids, twenty of which are commonly used. The present disclosure does not exclude the case that other substances (e.g., saccharides, lipids, and other modifications) are attached to the amino acid chains, and is not limited to the 20 amino acids that axe commonly used, either.

[0051] For a polypeptide sequence, a “conservative modification” includes a substitution, deletion, or addition of a polypeptide sequence, but does not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Eight sets of amino acids containing conservative substitutions are listed below: 1) alanine (A), glycine (G); 2) aspartate (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M). Tn some embodiments, the term “conservative sequence modification” is used to refer to an amino acid modification that does not significantly affect or change the target antigen-binding features of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant maintains a liquid condensate-forming efficiency of at least 80%, 85%, 90%, 95%, 98%, 99% or higher, e.g., 100-110% or higher, relative to a parental polypeptide.

[0052] Preferably, the difference in amino acids is conservative substitution, more preferably, one or more conservative substitutions.

[0053] As used herein, the term “conservative substitution” refers to amino acid substitutions which would not disadvantageous^ affect or change the expected properties of a protein / polypeptide comprising the amino acid sequence. For example, a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue physically or functionally similar (such as, having similar size, shape, charge, chemical property including the capability of forming covalent bond or hydrogen bond, etc.) to the corresponding amino acid residue. The families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (for example, lysine, arginine and histidine), amino acids having acidic side chains (for example, aspartic acid and glutamic acid), amino acids having uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having P-branched side chains (such as threonine, valine, isoleucine) and amino acids having aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine). Therefore, a corresponding amino acid residue is preferably substituted with another amino acid residue from the same side-chain family. Methods for identifying amino acid conservative substitutions are well known in the ait (see, for example, Brummell et al., Biochcm. 32: 1180-1187 (1993); Kobayashi ct al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0054] The twenty conventional amino acids covered herein are written to follow conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0055] In one embodiment, the liquid peptide condensate is a liquid-liquid phase separation (LLPS)- derived liquid peptide condensate. As used herein, “liquid-liquid phase separation” refers to a phenomenon in which a uniform mixture spontaneously divides into two liquid phases with different component concentrations. This phenomenon may occur by changing the pH, temperature, humidity, salt concentration or other suitable parameters of the environment. The active agent may form distinct boundaries that arc separated from the surrounding.

[0056] In some embodiments, the polypeptide can comprise one or more low-complexity amino acid sequences, which together can comprise a phase separation domain. Non-limiting examples of such phase separation domains are intrinsically disordered regions (IDR) and elastin-like polypeptide (ELP) domains. In response to one or more changes in the chemical or physical properties of the composition, non-limiting examples of which are composition, pH, temperature, volume, pressure, salt concentration, and the presence of one or more crowding agents, the phase separation domains of the polypeptide diffusing within the aqueous composition can spontaneously arrange themselves into an ordered structure, leading to the formation of a liquid condensate. Non-limiting examples of crowding agents include polyethylene glycol and dextran.

[0057] In some embodiments, the liquid condensate within an aqueous composition that also have a liquid internal phase can be referred to as “liquid-liquid phase separated (LLPS) droplets”.

[0058] In one embodiment, the L. acidophilus derived polypeptide forms the boundary' of the liquid condensate, separating the active agent from the surrounding environment. In some embodiments, the liquid condensate may comprise an L. acidophilus derived polypeptide, preferably a REP6 polypeptide, and may further comprise one or more components. In some embodiments, components encapsulated are probiotic microbe and / or pathogenic microbe. In some embodiments, the liquid condensate may encapsulate a mixture of two or more microbes. In some embodiments, the liquid condensate may encapsulate a nanoparticle, antibody, chemical or biological molecules, including but not limited to, vaccines, drug, antimicrobials and liposomes. In one embodiment, the nanoparticle is a nanoplastic. In one embodiment, the nanoparticle is a lipid nanoparticle (LNP).

[0059] Provided herein is the use of a polypeptide or the liquid peptide condensate as defined herein for aggregating probiotic microorganism for probiotic delivery. The term “probiotics” refers to live, non-pathogenic bacteria and fungus capable of producing beneficial effects on the health of a host. The term “probiotic component” refers to a preparation (e.g., a cell wall polysaccharide) derived from natural probiotics or engineered probiotics, or an analog of such a preparation. The term “analog” refers to a similar substance with similar activity to the preparation, comprising a derivative of the preparation (e.g., a derivative of a cell wall polysaccharide, which is capable of improving water solubility or activity), a synthetic product (e.g., a synthetic polysaccharide similar to a cell wall polysaccharide), other source preparations similar to the probiotic preparation (e.g., another source polysaccharide similar to a cell wall polysaccharide).

[0060] The term “pathogenic” refers to live, disease- or infection-causing bacteria and fungus capable of infecting a subject.

[0061] In some embodiments, the probiotic component is selected from: a preparation having a minimized bacterial immunogenicity and derived-from natural probiotics or engineered probiotics thereof, or engineered analogues of the preparation, wherein said preparation is preferably one or more selected from a group comprising: a probiotic water-soluble component, a probiotic semi-fluid-like component, a probiotic component water-insoluble particle, inactivated probiotics, and derivatives thereof.

[0062] Preferably, said probiotic water-soluble component is selected from a group comprising one or more of: a probiotic or disrupted probiotic supernatant component, a probiotic extract, a probiotic intracellular water-soluble component, water-soluble derivatives of water- soluble components or non-water-soluble components from probiotic cell wall components, and derivatives thereof; said probiotic semi-fluid-like component is preferably one or more selected from probiotic components whose aqueous mixture can form a semi-fluid-like composition, such as one or more of probiotic polysaccharides and analogues thereof; said probiotic component water-insoluble particle is one or more selected from a group comprising of: a disrupted probiotic precipitation component, a probiotic cell wall polysaccharide particle, a probiotic cell wall polysaccharide nanoparticle, and derivatives thereof; said inactivated probiotics is preferably selected from but not limited to inactivated probiotics having intact morphology; Wherein said probiotic water-soluble component is selected from a group comprising one or more of: a probiotic water-soluble p-glucan. preferably selected from a water-soluble (3-glucan of greater than 90% purity, a probiotic ribonucleic acid, and derivatives thereof.

[0063] In one embodiment, a probiotic component is a probiotic bacterium. In one embodiment, the probiotic bacterium is selected from a group comprising one or more of natural and / or engineered probiotic Bacillus, probiotic Lactobacillus, probiotic Bifidobacterium, probiotic Fungus. In one embodiment, the Bacillus comprises one or more selected from a group comprising Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, and Bacillus subtilis Natto. In one embodiment, the Lactobacillus comprises one or more selected from a group comprising Lactobacillus acidophilus, Lacticaseibacillus casei, Lactiplantibacillus plantarum, and Limosilactobacillus fermentum. In one embodiment, the Bifidobacterium comprises one or more selected from a group comprising Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Enterococcus faecalis, and Streptococcus faecalis. In one embodiment, the fungus comprises one or more selected from a group comprising yeasts and Brettanomyces bruxellensis, wherein the yeasts further comprise one or more selected from a group comprising Saccharomyces cerevisiae, Saccharomyces delbouillis, Saccharomyces Wickham, Saccharomyces boulardii, Pichia pastoris, Candida utilis, whey yeast.

[0064] The probiotic bacterium is selected from at least one of Bacillus, lactic acid bacteria, Bifidobacterium, and fungus, which may be natural-occurring or engineered. The Bacillus spp. such as, but not limited to, one or more of Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus megalerium, Bacillus firmus, Bacillus coagulans, Bacillus lentus, Bacillus pumilus, and Bacillus subtilis Natto. The lactic acid bacteria are Lactobacillus, wherein said Lactobacillus is selected from, but not limited to, at least one of L. acidophilus, Ligilactobacillus salivarius, Lacticaseibacillus casei, Lactiplantibacillus plantarum, and Limosilactobacillus fermentum.

[0065] The bifidobacteria include at least one of, but not limited to, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus helveticus, Enterococcus faecium, and Streptococcus faecalis. The fungi include yeasts and / or Brettanomyces bruxellensis, wherein said yeasts include at least one of, but not limited to, Saccharomyces cerevisiae, Torulaspora delbrueckii, Candida spp., Wickerhamomyces spp., Pichia spp., Torulopsis Candida, Saccharomyces chevalieri, Rhodotorula rubra, Schizosaccharomyces spp., Saccharomyces boulardii, Candida utilis and Saccharomyces boulardii.

[0066] In some embodiments, the probiotic bacteria include, but not limited to, Lactobacillus acidophilus, Limosilactobacillus fermentum, Lactobacillus helveticus, Limosilactobacillus reuteri, and Lactiplantibacillus plantarum. In one embodiment, a pathogenic component is a pathogenic bacterium. In some embodiments, the pathogenic bacteria include, but not limited to, Bacillus subtilis, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella, enterica, Enterococcus faecium, Escherichia, coli, Acinetobacter baumannii, Clostridium difficile, and Staphylococcus aureus.

[0067] The term “inactivated probiotics” refers to the preparation obtained after inactivation treatment such as high temperature inactivation, high temperature and high-pressure inactivation, ultraviolet inactivation, chemical reagent inactivation, radiation inactivation, sonication inactivation, etc.

[0068] In some embodiments, the liquid condensate of this invention is to be used to deliver a pathogenic bacterium. The purpose of delivering a pathogenic bacterium to a subject includes, but not limited to, vaccination, anti-canccr therapy, antigen delivery, and as delivery vectors. In one embodiment, the pathogenic bacterium is a live-attenuated or dead bacterium.

[0069] The term “live-attenuated bacterium” refers to a strain of bacterium that has been weakened, and may produce a broad immune response in a subject. In one embodiment, the liquid condensate is to be used to deliver a live-attenuated or dead bacterium that may act as a vaccine to trigger an immune response in a subject. The term “immune response” refers to the activation of immune cells, including but not limited to B cells and T cells in the immune system to produce antibodies or activate cell-mediated immunity in a subject.

[0070] The term “live-attenuated pathogenic bacterium” refers to a strain of disease-causing bacterium in which the virulence or infectivity has been weakened or weakened to a non- pathogenic level. In one embodiment, the liquid condensate is to be used to deliver a live- attenuated pathogenic bacterium that may act as a vaccine to trigger an immune response in a subject. In some embodiments, the liquid condensate of this invention may form uniform particles. In some embodiments, the diameters of spherical particles of condensate compounds may have a distribution with an average of less than 1000 micrometers (pm). The condensate compounds of this invention can provide their own multicomponent formulations. In some embodiments, a compound can be combined with other agents for drug delivery such as carries or vehicles for delivery to a cell, or a subject, or various delivery matrices, for in vivo therapeutics.

[0071] In one embodiment, the liquid condensate of this invention may be used to separate, fractionate, concentrate, purify, and remove compounds, molecules, particles and cells in the aqueous surrounding environment.

[0072] Composition

[0073] The microbial to liquid condensates ratio is kept at 25% in the examples provided and it can be increased to 35% or more as the concentration of the polypeptide used in the formation of liquid condensates increases.

[0074] Provided herein is a composition comprising i) a polypeptide or a liquid peptide condensate as defined herein and ii) a microorganism.

[0075] In one embodiment, the composition comprises a pharmaceutically acceptable carrier.

[0076] By “pharmaceutically acceptable carrier” is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.c., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, colouring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

[0077] Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient(s), its use in the pharmaceutical compositions is contemplated.

[0078] The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Suitable pharmaceutical compositions may be administered orally, topically, intravenously, subcutaneously or intramuscularly. In some embodiments, the compositions are in the form of injectable or infusible solutions. In some embodiments, the administration is parenteral e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intranasal, topical or transdermal). Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in a micro-emulsion, liposome, or other ordered structure suitable to high ding concentration.

[0079] The pharmaceutical composition may be a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. hi some embodiments, there is provided a method for the delivery of an active agent, comprising: (i) providing a composition of a peptide liquid condensate as described herein; and (ii) exposing the peptide liquid condensate to conditions that trigger the release of the active agent from therefrom.

[0080] In some embodiments, the conditions that trigger the release of the active agent are selected from the group consisting of pH changes, ionic strength, guest molecule, exposure to light (UV irradiation), exposure to release agents, and combinations thereof. In some embodiments, there is provided a method for treating disease in a subject in need thereof, comprising: (i) administering a composition comprising a peptide liquid droplet as described herein; and (ii) exposing the peptide liquid droplet to conditions that trigger the release of the pharmaceutical or diagnostic agent.

[0081] In some embodiments, the conditions that trigger the release of the pharmaceutical or diagnostic agent are selected from the group consisting of pH changes, exposure to release agents, and combinations thereof.

[0082] Accordingly, under encapsulation-permissive conditions, an active agent may easily be captured from the surrounding of the compartment by diffusion, and under release- permissive conditions the active agent may be similarly easily released by diffusing out of the peptide liquid condensate. This allows a spontaneous capture and release of active agent, depending on the surrounding conditions. It is further noted that there is no requirement for the compartment to fuse with cells in order to deliver active agent. A further advantage of the permeability and “spontaneity” of the liquid condensate is that it may capture or release an active agent in response to a change in environmental conditions, or stimuli. The compartments of the invention may be used, for example, for delivery of molecules to the extracellular matrix (possibly for enzyme replacement), for local (topical) delivery of active agents, or for capturing access molecules.

[0083] The term “compartment”, as used herein, relates to micron-scale, liquid-like, membraneless body or assembly, also referred to as a liquid condensate. Such compartments exist in mammalian cells and usually include proteins and nucleic acids. They are capable of compartmentalizing reactions such as viral replication and assembly of viral particles, and arc therefore capable of trapping various molecules.

[0084] In some embodiments, the solution may contain an inorganic or organic salt. For example, the aqueous solution may contain sodium chloride at a concentration of less than or equal to about 1 M, less than or equal to about 0.5 M, and less than or equal to about 0.25 M.

[0085] In some embodiments, the peptide liquid condensate compounds of a particular' distribution of sizes can be isolated from the solution. In one embodiment, the solution containing the liquid condensate encapsulating active agent is dialyzed to remove excess or unbound peptide components. Tn some embodiments, isolated peptide liquid condensates are lyophilized.

[0086] In one embodiment, the polypeptide as defined herein is an isolated polypeptide.

[0087] As used herein, an “isolated” protein or polypeptide (e.g., the protein liquid condensate of the present invention) is a protein or polypeptide that has been isolated from components of its natural environment. In some embodiments, the protein or polypeptide is purified to more than 80%, 90%, 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (1EF), capillary electrophoresis), or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0088] The terms “active agent” and “payload” may be used interchangeably, which refers to any components that are encapsulated by the liquid condensate that comprises chemical or biological in nature that is intended to be delivered to the target sites of a subject. The said components include but are not limited to nucleic acids, peptides, lipids, charged molecules, ions, microbes, microbials, viruses, enzymes, prebiotics, vaccines, bacteriocins and nanoparticles.

[0089] In one embodiment, the composition as defined herein for use as a nutraceutical, supplement and / or ingredient. In some embodiments, the composition is to be delivered to the site of interest of a subject to enable beneficial functions and / or outcomes to the host. The beneficial functions and / or outcomes include but are not limited to skin health, ocular health, respiratory health, metabolic health, neurological axis, immunomodulation and female health, via gut-organ axis.

[0090] Additionally, REP6 has the ability to act as a carrier for chemical entities, and the delivery to site of interest, enables beneficial host functions, such as skin health, ocular health, respiratory health, metabolic health, neurological axis, immunomodulation and female health, via gut-organ axis.

[0091] Treatment Provided herein is a composition as defined herein for use as a medicament.

[0092] Provided herein is a method of treating and / or preventing a mucosal, epithelial and / or gut condition in a subject, the method comprising administering a composition as defined herein to the subject.

[0093] The terms “patient”, “subject”, “host” or “individual” used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (c.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcincs (c.g., pigs), equines (c.g., horses), canines (c.g., dogs), felines (c.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish.

[0094] The terms “treating”, “treatment” and the like include relieving, reducing, alleviating, ameliorating or otherwise inhibiting the effects of the metabolic disease for at least a period of time. It is also to be understood that terms “treating”, “treatment” and the like do not imply that the disease, or a symptom thereof, is permanently relieved, reduced, alleviated, ameliorated or otherwise inhibited and therefore also encompasses the temporary relief, reduction, alleviation, amelioration or otherwise inhibition of the disease, or of a symptom thereof.

[0095] As used herein, the terms “gut condition”, “gut disorder”, and “gut disease” may be used interchangeably, and refers to a group of acute and / or chronic disorders related to the gastrointestinal system of a subject, including the oral cavity, esophagus, stomach, liver, pancreas, gall bladder, small intestine, large intestine, rectum and anus. Gut conditions or gut disease include but not limited to stomach pain, constipation, bloating, diarrhoea, hemorrhoids, colon polyps, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), Crohn’s disease, colitis, ulcerative colitis, gastritis, diverticulitis, gastroesophageal reflux disease (GERD), celiac disease, lactose intolerance, gastroenteritis, food poisoning, and Helicobacter pylori infection.

[0096] As used herein, the terms “mucosal condition”, “mucosal disorder”, and “mucosal disease” may be used interchangeably, and refers to a condition affecting the mucous membrane in a subject, such as the lining of mouth, nose, eyes, genitals and digestive tract. The condition may be an infection caused by bacteria, viruses and fungus. The condition may be an autoimmune condition wherein the immune cells or system of a subject attacks cells, tissues or organs of the same subject. Non-limiting examples of mucosal condition includes Herpes infection, Candidiasis infection, Lichen Planus infection, oral mucositis and canker sores.

[0097] As used herein, the terms “epithelial condition”, “epithelial disorder”, and “epithelial disease” may be used interchangeably, and refers to a condition affecting the epithelial lining of organs and surfaces of a subject. Non-limiting examples of epithelial conditions include eczema, psoriasis, skin inflammation, skin rashes, epithelial carcinoma, asthma, dry skin, scaly skin, hepatitis, autoimmune hepatitis, ulcerative colitis and Crohn’s disease.

[0098] In one embodiment, there is also provided a method of treating a gut-organ axis condition, the method comprising administering a composition as defined herein to the subject.

[0099] As used herein, the terms “gut-organ axis condition”, “gut-organ axis disorder”, “gut-organ disease” and “disease of the gut-organ axis” may be used interchangeably, and refers to a condition affecting the bidirectional communication of the gut in a subject and with the organs of the same subject, such as the brain, liver, kidney and skin. Non-limiting examples of the condition includes Alzheimer’s disease, Parkinson’s disease, schizophrenia, depression, mood disorders, anxiety, diabetes, obesity, irritable bowel syndrome (IBS), constipation, dyspepsia, gastroparesis, liver injury, kidney injury and chronic obstructive pulmonary disease (COPD).

[0100] Other uses As used herein, the term “skin, respiratory and mucosal surfaces” refers to area where treatment can be directly or indirectly applied to the affected area. The treatment includes but not limited to a topical cream on skin for therapeutic purposes or for cosmetic purposes such as to improve skin texture or hydration; nasal spray for respiratory tract or suspended liquid formulation for gastrointestinal mucosa.

[0101] As used herein, the term “nanoparticle” refers to particles having a particle size on the nanometer scale, less than 1 micrometer. For example, the nanoparticle may have a particle size up to about 50 nm. In another example, the nanoparticle may have a particle size up to about 40 nm. In another example, the nanoparticle may have a particle size up to about 30 nm. In another example, the nanoparticle may have a particle size up to about 20 nm. In another example, the nanoparticle may have a particle size up to about 10 nm. In another example, the nanoparticle may have a particle size up to about 6 nm. In one embodiment, the nanoparticles have a diameter range of between 1-4 nm, 2-6 nm, 3-7 nm, 4-8 nm, 5-9 nm, 6-10 nm, 7-1 1 nm, 8-12 nm, 9-13 nm, 10-14 nm 1 1-15 nm, 12-16 nm, 13-17 nm, 14-18 nm, 15-19 nm or 16-20 nm.

[0102] By “antibody” is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and nonimmunoglobulin derived protein frameworks that exhibit antigen-binding activity. Representative antigen-binding molecules that are useful in the practice of the present invention include polyclonal and monoclonal antibodies as well as their fragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies), and fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding / rccognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.

[0103] As used herein, the term “recombinant” refers to the production of a polypeptide or protein by a biological host. The host may be selected from, including but not limited to, a mammalian expression system, an insect cell expression system, a yeast expression system, and a bacterial expression system. In one embodiment, the polypeptide / protein of the present invention is arccombinant poly peptide / protein expressed in a prokaryotic organism (c.g., an Escherichia coli cell) or a eukaryotic host cell (e.g., a mammalian host cell). In the present invention, the liquid condensate has binding affinity for target surfaces. Nonlimiting examples of target surfaces including epithelial mucosa, lipid, mucin, cell surfaces, artificial cell surfaces, and glass. In the present invention, the affinity of the liquid condensate may be modified by adjusting different parameters. Non-limiting examples of adjustable parameters include concentration of polypeptide, concentration of components in the environment of the polypeptide, pH, temperature, humidity, salt concentration, types and concentrations of microbes, and use of surfactant. Non-limiting surfactants include polysorbates, sucrose esters, lecithin, glycerol esters of wood rosin (GEWR), sorbitan esters, sodium stearoyl lactylate, propylene glycol esters, calcium stearoyl-2-lactylate, sodium caseinate, glycerol monostearate, polyglycerol esters and polyethylene glycol.

[0104] As used herein, “affinity” or “binding affinity” refers to the intrinsic binding affinity that reflects interactions between members of a binding pair. The affinity of a molecule X for its partner Y may typically be represented by a dissociation constant (KD), and the dissociation constant is a ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively). Affinity may be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0105] As used herein, the term “about” when used in conjunction with a numerical value is meant to encompass numerical values within the range of the lower limit that is 5% less than the specified numerical value and the upper limit that is 5% greater than the specified numerical value.

[0106] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0107] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0108] Throughout this specification and the statements which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0109] Throughout this specification and the statements which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0110] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0111] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary' skill in the art to which this invention belongs.

[0113] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0114] EXAMPLES EXAMPLE 1

[0115] Liquid condensates of a cell-derived short peptide sequence of surface (S-) layer protein from Lactobacillus cells

[0116] The full-length S-layer protein of L. acidophilus ATCC4356 or PCB 001, accession number: KHE30826.1, was recombinantly expressed as several peptide sequences, REP1, REP2, REP4, REP5, REP6 and REP6-GFP (Table 1). REP6-GFP refers to a REP6 peptide that is conjugated to a green fluorescent protein (GFP) for visualization purposes. The condensate forming property of the peptides as well as the full-length protein was examined. Among the investigated peptides, REP6 (276 amino acid residues), exhibited the highest propensity to undergo phase separation, thus forming liquid condensates. These condensates coalesced and demonstrated affinity for surfaces, including glass (Figure 1). REP6 will be used as an example to demonstrate the various functions of the liquid condensates. It is worth noting that REP6 was harvested from L. acidophilus ATCC 4356, but it is expected to occur in other Lactobacillus species and not limited to this species and strain.

[0117] Table 1 : Tabulation of amino sequences representing six novel peptides (REP1 , REP2, REP4, REP5, REP6 and REP6-GFP) of L. acidophilus ATCC 4356 / PCB 001, accession number: KHE30826.1.

[0118] EXAMPLE 2 Liquid condensates display binding affinity for microorganisms and lipid membranes

[0119] The binding affinity of the liquid condensates for bacterial cells was verified using twelve different strains of bacteria, including five Lactobacillus strains known for probiotic potential (L. acidophilus PCB 001, L. fermentum PCI, L. helveticus PCB 009, L. reuteri PCB 004 and L. plantarum PCB 011), two non-lactic acid strains (Pseudomonas aeruginosa and Bacillus sublilis) and five pathogenic bacteria (Escherichia coli, Salmonella enterica, Listeria monocytogenes, Clostridium difficile and Acinetobacter baumannii) (Figure 2). These liquid condensates effectively facilitate the aggregation of bacteria in both single- and mixed-species cultures (Figure 2A-B and 2E-H, and 2K-O). The physicochemical conditions of the solution are important as shown by the lack of binding of the two non-lactic acid bacteria to REP6 liquid condensates under certain environmental conditions, such as slightly acidic to neutral pH (i.e., pH 6-7) (Figure 2I-J).

[0120] Nonetheless, the reduction of the pH in the mixture of the bacterial cells and the liquid condensate enabled the binding of the otherwise non- aggregating Pseudomonas aeruginosa cells to the surface of liquid condensates (Figure 3 A). This identifies an approach to regulate the association and release of specific microorganisms based on the pH of the site of interest. Furthermore, the direct coating of liquid condensates onto lipid membranes indicates strong affinity of the liquid condensates to cellular surfaces (Figure 3B and 3C) and thus facilitate the delivery of bacterial cell-liquid condensate mixture to cellular surface such as gut epithelial lining. Moreover, addition of polypeptide to lactic acid bacteria can induce peptide liquid condensate formation and results in aggregation of bacteria (Figure 3D).

[0121] Additionally, it was shown that nanoparticles (e.g., 50 nm polystyrene nanoplastics) and biological molecules (e.g., antibody) bind to the liquid condensates in a manner similar to microbial cells (Figure 3E and 3F respectively). Hence, this demonstrates the application of REP6 to include inert particles and molecules and the potential of delivering and / or sequestering these materials using peptide liquid condensates.

[0122] EXAMPLE 3

[0123] Lactobacillus cells aggregated with REP6 liquid condensates maintained viability during overnight storage at 4°C and simulated gastrointestinal environment The impact of REP6 liquid condensates on the viability of the aggregated Lactobacillus cells was validated. This was done by comparing the viability of cells that had been stored overnight at 4°C with or without REP6 (Figure 4A). The viability of the Lactobacillus cells remained constant at 108colony forming units (CFUs) under all tested conditions. Similarly, subjecting aggregated Lactobacillus cells to simulated gastric and intestinal fluids (i.e., buffer in the presence of pepsin at low (i.e., pH 2) and high pH (i.e., pH 7) respectively), showed pH independent effect on cell viability and aggregability in both conditions (Figure 4B-E).

[0124] These results highlight the non-toxic nature of the liquid condensates and suggest that REP6 docs not induce microbial cell death following overnight storing at 4°C or under a change in the environmental pH.

[0125] EXAMPLE 4

[0126] REP6 liquid condensates enabled the delivery of Lactobacillus bacteria as microbial aggregates with enhanced cellular adhesion to in vitro gastrointestinal epithelium mucus layer

[0127] Microscopy observation demonstrated REP6 facilitated adhesion of microbes onto mucin surface as cellular aggregates, following incubation of premixed REP6 liquid condensate- microbial aggregates to Porcine Type III mucin layer (Figure 5). The distinct cellular aggregation deposition onto mucin layer was not observed in the absence of REP6 and the cells attached to the mucin surface either as a single cell or in pairs (Figure 5A).

[0128] Collectively, no significant effect of REP6 on the viability of lactic acid producing cells (i.e., Lactobacillus cell) in the stomach and intestinal conditions along with the enhanced mucin adhesion properties in cellular aggregates demonstrate the feasibility of delivering bacteria, preferably bacterial cells with probiotic potential, in the form of microcolonies / aggregates to the site of interest with strengthened cellular colonization properties. Furthermore, the incorporation of chemical or biological molecules, such as antibody, in the preparation could tentatively enhance its functionality while delivering microbial cells of interest.

[0129] EXAMPLE 5

[0130] (i) A delivery vehicle for bacterial cells using liquid condensates of a peptide derived from Lactobacillus S-layer protein

[0131] Delivery of probiotics

[0132] The strong cellular association capabilities of liquid condensates to various beneficial bacteria will facilitate the delivery and attachment of an optimal probiotic cell preparation to the site of interest. This preparation aims to create a healthy gut microbial community and also boost beneficial biofilm formation in the gut.

[0133] Gut microbiome modulation

[0134] The affinity of liquid condensates for pathogenic bacteria enables better association of the condensates with pathogens at a site of infection. The delivery of liquid condensate-probiotic bacterial aggregates to the target site will facilitate the inhibition / replacement of gut bacterial pathogens. The efficiency of the liquid condensate-bacteria aggregates can be further improved / enhanced by delivering probiotic bacterial cells along with chemical or biological molecules (e.g., prebiotics for beneficial bacteria growth or antimicrobials drugs, bacteriocins and nanoparticles that can act specifically on pathogens) within the condensates. Additionally, the liquid condensates can be used to facilitate the delivery to the targeted site of microorganisms that inherently exhibit lower adhesion properties, e.g., Bifidobacterium.

[0135] This invention also enables the administration of beneficial microbes in the form of microbial aggregate by utilizing the high specificity of REP6 liquid condensates to the microbial species and affinity to surfaces including artificial cell surfaces and gut lining. Furthermore, the comparable count of colony forming units (CFUs) detected in the liquid condensate-cell aggregates stored at 4°C overnight as well as in the simulated gastric juice and intestinal fluids provide further evidence that liquid condensates arc a suitable medium for delivering probiotic bacteria as well as, chemical and biological molecules. The microbial cell -aggregating feature of REP6 liquid condensate to microbes, together with pH tunable association and enhanced surface adhesion properties, enables the deliver}' of a customizable probiotic preparation to create, restore and sustain an optimum gut microbiome in the host. ii) Naturally derived REP6 liquid condensates can act as carrier for the delivery of chemical and biological entities including drugs, vaccines, antimicrobial agents

[0136] The associating and encapsulating properties of REP6 to a wide range of chemical and biological entities such as nanoparticles and antibodies, demonstrated its potential as a carrier for these compounds, which separate the compounds from the surrounding environment. Correspondingly, the affinity of REP6 liquid condensates to surfaces suggests improved delivery of the associated materials to the site of interest. The ability to act as a carrier for chemical entities, and the delivery to site of interest, enables beneficial host functions, such as skin health, ocular health, respiratory health, metabolic health, neurological axis, immunomodulation and female health, via gut-organ axis. iii) In situ separation, fractionation, concentration, purification and removal of compounds, molecules, particles and cells using REP6 liquid condensates

[0137] The associating and encapsulating properties of REP6 to a wide range of chemical and biological entities such as nanoparticles strongly suggest the properties of REP6 for aggregation and subsequent separation of these compounds and particles from their original matrices. Hence, REP6 liquid condensates can be utilized as a tool / medium for in situ separation or concentration of targeted molecules or compounds in a mixture where chemical-based methods arc not applicable.

Claims

1. CLAIMS1. A polypeptide comprising or consisting of an amino acid sequence having at least 70% (including 80%, 90%, 95% or 100%) sequence identity to SEQ ID NO: 1, or a fragment thereof, wherein the polypeptide, or fragment thereof, is capable of forming a liquid peptide condensate.

2. The polypeptide of claim 1, wherein the polypeptide does not comprise an amino acid sequence having at least 70% (including 80%, 90%, 95% or 100%) sequence identity to SEQ ID NO: 9.

3. The polypeptide of claim 1 or 2, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO: 1.

4. A liquid peptide condensate, comprising a polypeptide of any one of claims 1 to 3.

5. The liquid peptide condensate of claim 4, wherein the liquid peptide condensate is a liquid-liquid phase separation (LLPS)-derived liquid peptide condensate.

6. The liquid peptide condensate of claims 4 or 5, wherein the diameter of the liquid peptide condensate is less than 1000 micrometers (pm).

7. The liquid peptide condensate of any one of claims 4 to 6, wherein the liquid peptide condensate aggregate and / or encapsulates components from the surrounding aqueous solution at about pH 3.0 to about pH 9.0.

8. A composition comprising i) a polypeptide of any one of claims 1 to 3 or a liquid peptide condensate of any one of claims 4 to 7 and ii) a microorganism.

9. A composition comprising i) a polypeptide of any one of claims 1 to 3 or a liquid peptide condensate of any one of claims 4 to 7 and a suitable functional biological or chemical agent, wherein the agent is selected from a group comprising a drug, a vaccine, a small molecule, aprebiotic, a nanoparticle, a bacteriocin, an antibody, and a combination thereof.

10. The composition of claims 8 or 9, wherein the composition comprises a pharmaceutically acceptable carrier.

11. The composition of any one of claims 8 to 10 for use as a medicament.

12. The composition of any one of claims 8 to 10 for use as a nutraceutical and / or supplement.

13. Use of a polypeptide of any one of claims 1 to 3 or the liquid peptide condensate of any one of claims 4 to 7 for aggregating and / or encapsulating probiotic microorganism for probiotic delivery.

14. Use of a polypeptide of any one of claims 1 to 3 or the liquid peptide condensate of any one of claims 4 to 7 for aggregating, encapsulating, adhering and / or separating pathogenic microorganism.

15. Use of polypeptide of any one of claims 1 to 3 or the liquid peptide condensate of any one of claims 4 to 7 for delivering a suitable functional biological or chemical agent, wherein the agent is selected from a group comprising a drug, a vaccine, a small molecule, a probiotic, a nanoparticlc, a bactcriocin, an antibody, and a combination thereof.

16. A method of treating and / or preventing a mucosal, epithelial, and / or gut condition or disease in a subject, the method comprising administering a composition of any one of claims 9 to 11 to the subject.

17. The method of claim 16, wherein the gut condition or disease is stomach pain, stomach bloating, stomach gas, diarrhoea, colitis, Crohn’s disease and / or irritable bowel syndromes (IBS).

18. The method of claim 16, wherein the mucosal condition or disease is Herpes infection, Candidiasis infection, Lichen Planus infection, oral mucositis and / or canker sores.

19. The method of claim 16, wherein the epithelial condition or disease is eczema, psoriasis, skin inflammation, skin rashes, epithelial carcinoma, asthma, dry skin, scaly skin, hepatitis, autoimmune hepatitis, ulcerative colitis and / or Crohn’s disease.

20. A composition of any one of claims 9 to 11 for use in treating and / or preventing a mucosal, epithelial, and / or gut condition or disease in a subject.

21. Use of a composition of any one of claims 9 to 11 in the manufacture of a medicament for treating and / or preventing a mucosal, epithelial and / or gut condition or disease in a subject.

22. A method of producing a liquid peptide condensate, wherein the method comprises (i) isolating the polypeptide of claim 1; (ii) contacting the polypeptide of claim 1 with a suitable aqueous solution; and (iii) adjusting the pH to suitable range.

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