Compositions containing probiotic supernatants

Abstract

The present invention is directed to drink or food compositions or formulations containing components of culture supernatants from probiotic microorganisms such as bacteria and fungi. The present invention also relates to methods for making and using said compositions, such as a dietary supplement, a medication or a stimulant.

Description

[0001] COMPOSITIONS CONTAINING PROBIOTIC SUPERNATANTS

[0002] TECHNICAL FIELD

[0003] The present invention is directed to drink or food compositions or formulations containing components of culture supernatants from probiotic microorganisms such as bacteria and fungi. The present invention also relates to methods for making and using said compositions, such as a dietary supplement, a medication or a stimulant.

[0004] In particular, the present invention relates to drink and food compositions or formulations that include secreted molecules, molecules shed from the surface of probiotic microorganisms, and water from a supernatant component which corresponds to cell-free portion of a probiotic culture. The compositions or supernatants alone (secreted or shed molecules, with or without water) may be useful for a variety of applications such as improved hydration, metabolism and wellbeing of consumers, and aiding in recycling a water-rich by-product of the probiotic industry (supernatants). However, it will be appreciated that the invention is not limited to these particular fields of use.

[0005] BACKGROUND

[0006] BACKGROUND TO WATER WASTE

[0007] The following discussion of the prior art is provided to place the invention in an appropriate technical context and enable the advantages of it to be more fully understood. It should be appreciated, however, that any discussion of the prior art throughout the specification should not be considered as an express or implied admission that such prior art is widely known or forms part of the common general knowledge in the field in Australia or any other jurisdiction or that this prior art could reasonably expected to be combined by a person skilled in the art..

[0008] The Earth’s surface is made of 70% water, although only 3% of this corresponds to fresh water, with another 65% sitting frozen on mountain tops and polar areas. In fact, the amount of water available for drinking, farming, personal hygiene and services is so rare, that water scarcity has become a major reason for concern. To this end, international agencies have warned that over 65% of the globe could experience water shortages by 2025. To aggravate this situation, the world’s population continues to grow at alarming rates, and surrounding ecosystems become stressed by rapidly changing weather patterns, whereby once fertile areas now experience constant water shortage and drought. Globally, 1.1 billion people currently lack access to clean water, while 2.7 billion people experience water scarcity for at least one month of every year. This scenario creates a plethora of issues, ranging from malnutrition to social injustice, and from conflicts to disease occurrence, the later which affects up to 2.4 billion people worldwide and causes the annual death of at least one million people (mostly children).

[0009] Accordingly, there exists a need for improved ways of recycling waste water into consumable products.

[0010] BACKGROUND TO PROBIOTIC CELLS AND MOLECULES

[0011] Microbial probiotics can be consumed through fermented foods or supplements, whereby their main functional role is to restore and maintain the balance of the gut microbiome in humans and animals. This has been indeed linked to a range of health benefits, while their imbalance or absence starts to be more widely associated with the onset of disease human and animal diseases.

[0012] Globally, probiotic consumption occurs through foods, drinks, supplements, and the industry now studies the possibility of using therapeutic probiotics as medications supported by scientific evidences that allow strong clinical claims. Bearing this in mind, the probiotic industry aims to intensify and diversify the commercialisation of probiotic mixtures, which has been traditionally occurred as living probiotic cells or non-living and non-replicating probiotic extracts.

[0013] Probiotic cells have been widely associated with benefits including weight loss, digestive health, immune and endocrine function and more, and literature mining has revealed that key health benefits linked to probiotics include:

[0014] 1) support towards ensuring and / or restoring the balance of beneficial bacteria in the digestive system of humans and animals, whereby an imbalance means that the gut or organs and tissues are mostly colonised and / or populated by pathogenic microbes and their molecules, as a result of illness, antibiotic intake, poor diet and more.

[0015] 2) assistance in preventing and treating diarrhea, as well as reducing its severity as a common side effect of taking antibiotics that promote gut imbalance. In addition, studies have found that probiotics also help with other forms of diarrhea not associated with antibiotics, for example caused by infections, travellers’ diarrhea or diarrhea caused by other factors among children and adults.

[0016] 3) improvement of certain mental health conditions, due to their direct link to gut health which has been demonstrated through both animal and human studies. In fact, human supplementation with probiotics strains for as little as 1 month was found to contribute to improve anxiety, depression, autism, obsessive-compulsive disorder (OCD) and memory loss. Additionally, another study conducted among chemical workers for 6 weeks found that those who consumed daily probiotic yogurts (or took a daily probiotic capsule) experienced general health benefits, reduced depression and anxiety, and stress alleviation.

[0017] 4) certain probiotics can promote cardiac benefits, for example by lowering LDL levels, reducing cholesterol and blood pressure, all which have been shown to be achieved when lactic acid-producing bacteria were found to break down bile in the gut and preventing it from being reabsorbed in the gut and blood (as cholesterol).

[0018] 5) reduction of the severity of certain allergies and eczema, particularly among children and infants fed probiotic-supplemented milk (compared to infants fed milk without probiotics). Additionally, women who take probiotics during pregnancy are known for delivering children with over 80% less risk of developing eczema in the first two years of life, while specific probiotic species were also proven to reduce inflammatory responses in people with milk and / or dairy allergies.

[0019] 6) The immune system of humans and animals can also be boosted by the presence of probiotics, both by inhibiting the growth of harmful gut bacteria, and by also serving as stimulatory mechanisms to modulate immune responses and induce IgA-producing cells, T lymphocytes and natural killer cells. This has in fact been linked to a reduction in the likelihood and duration of respiratory infections, as well as the risk of urinary tract infections (UTIs) in women.

[0020] 7) Finally, probiotics help the treated body burn more fat, or prevent its accumulation. This occurs through prevention of the absorption of dietary fat in the intestine, which is then excreted through feces rather than stored in the body. Probiotics are also known for helping consumers space out their food consumption for longer periods, resulting in more calories being burned, while less are ingested resulting in the storage of less fat. Of note, this is partly caused by hormonal up-regulation (e.g. GLP-1 in humans).

[0021] Despite all evidences, there are no studies or commercial products that specifically address the secretome of probiotics, which we herein denominate supernatants, or postbiotic functional mixtures, or water-rich component depleted from probiotic cells.

[0022] BACKGROUND TO DIETARY PROTEINS

[0023] Since early 2000s, the muscle-related benefits achieved by consuming high- protein diets have become increasingly clear in the literature. Despite this, dietary protein consumption patterns have remained relatively unchanged among American, European and Australian adults. This may be due to a number of factors, such as:

[0024] • misconceptions related to high-protein diets,

[0025] • incorrect / inefficient consumption,

[0026] • low-quality proteins, and

[0027] • incorrect perception about detrimental effects.

[0028] Global markets have confirmed a desire for increased protein supply, whereby traditional sources include animal and plant proteins. However, both protein origins have been associated with issues related to greenhouse gas emissions, water consumption, land use, environmental reliance, and other factors that prevent sustainability. Additionally, diets purely based on plant proteins are known to reduce the intake of essential amino acids (in comparison to animal proteins), which ultimately reduces protein synthesis in the body and increases the risk of hypoproteinemia, due to a reduced intake in methionine and lysine.

[0029] New technologies being considered include the consumption of insects and laboratory meats, which require further economic optimisation and scientific validation. Additionally, fungal proteins have been commercially trailed for two decades (e.g. Korn foods), but the amount of fungal alternative protein used in pre-packed meals ranges between 15 and 43%, meaning that most of the global protein supply still depends on farming systems.

[0030] It is an object of the present invention to overcome or ameliorate one or more the disadvantages of the prior art, or at least to provide a useful alternative.

[0031] SUMMARY

[0032] The present invention is directed to drink or food compositions or formulations containing components of culture supernatants from cellular organisms such as prokaryotic or eukaryotic cultures. Also disclosed are methods for making and using said compositions, such as a dietary supplement, a medication or a stimulant.

[0033] Accordingly, in a broad form, the present invention relates to a composition comprising: secreted molecules and shed molecules and / or probiotic cell structures that do not constitute the living cell, wherein these are at least partly derived from at least one culture supernatant from a cellular organism; and water, wherein the water is at least partly derived from at least one culture supernatant from at least one cellular organism; and a base.

[0034] As used herein cellular organism as used herein refers to all cellular organisms and includes but is not limited to mammalian cells, insect cells, plant cells, bacterial cells, fungal cells and any prokaryotic or eukaryotic cultures, including multi-cellular cultures, multi- cell type cultures, multi-species cultures and polymicrobial cultures.

[0035] In another aspect the invention relates to a composition, such as a drink or food composition, comprising water that has been at least partly derived from at least one culture supernatant from at least one probiotic microorganism and a base.

[0036] In a first aspect, the invention resides in a composition comprising: water, wherein the water is at least partly derived from at least one culture supernatant from at least one probiotic microorganism; and a base.

[0037] Suitably, the base comprises a protein source, a carbohydrate source, a lipid source and / or a sweetening agent.

[0038] In some embodiments, the protein source is or comprises a non-animal-derived protein source. In certain embodiments, the carbohydrate source is or comprises a non- animal-derived carbohydrate source. In various embodiments, the lipid source is or comprises a non-animal-derived lipid source. To this end, the protein source, the carbohydrate source and / or the lipid source are suitably derived at least in part from a plant. More particularly, the protein source, the carbohydrate source and / or the lipid source may be derived at least in part from: a plant extract; a fruit extract, such as a fruit powder or a fruit juice; a flower extract, such as a flower powder; and / or a seed extract, such as a seed powder, a seed butter, or seed oil (e.g., essential oils, flavour oils).

[0039] Suitably, the protein source, the carbohydrate source and / or the lipid source are selected from the group consisting of cocoa powder, cacao powder, whey powder, milk powder, cacao butter, vegetable oils, coconut milk, lemon powder, honey powder, orange powder, berry powder, hibiscus powder, native Australian bush tucker (fruits and flowers) powder, tropical fruit and flower powder, and any combination thereof, whereby powder ingredients may be replaced by puree, mash, extracts, essences, liquors, liquids, juices and / or concentrates.

[0040] In some embodiments, the composition is a substantially vegetarian and / or vegan composition. To this end, the composition may be suitably substantially free of animal- derived components. Suitably, the composition comprises from about 0.1 wt% to about 99 wt% of the at least one culture supernatant from the at least one probiotic microorganism.

[0041] Suitably, the composition comprises from about 0.1 wt% to about 99 wt% of the base.

[0042] Suitably, the composition comprises from about 0.1 wt% to about 99 wt% of the protein source, the carbohydrate source and / or the lipid source.

[0043] Suitably, the composition comprises from about 0.1 wt% to about 30 wt% of the sweetening agent.

[0044] In some embodiments, the composition further comprises one or more of a pH modifier, a preservative, a solvent, an emulsion stabilizer, a colouring agent, a flavouring agent, a vitamin and a mineral.

[0045] In certain embodiments, the composition and / or supernatants and / or supernatant molecules are in the form of or for use as a supplement. More particularly, the composition can be a protein or a vitamin supplement or a carboxylic acid supplement, or an amide supplement or an amine supplement or other products containing the composition and / or supernatants and / or supernatant molecules.

[0046] In certain embodiments, the composition and / or supernatants and / or supernatant molecules are in the form of or for use as a medicament or stimulant. More particularly, the composition can stimulate the expression of genes or alter the production of hormones or change the molecular profile within the consumer’s body.

[0047] In certain embodiments, the composition and / or supernatants and / or supernatant molecules are in the form of or for use as an ingredient. More particularly, the composition can contain soft or clinical claims proven by scientific evidences and literature mining.

[0048] In certain embodiments, the composition and / or supernatants and / or supernatant molecules are in the form of or for use as an additive or a processing aid. More particularly, the composition can extend the shelf-life of a food, drink, beverage, medication, cosmetics and other products thereof containing the composition and / or supernatants and / or supernatant molecules.

[0049] In various embodiments, the composition is substantially gluten free.

[0050] In particular embodiments, the composition is substantially lactose free.

[0051] In a preferred embodiment, the composition according to this aspect of the present invention comprises: secreted molecules and shed molecules and / or probiotic cell structures that do not constitute the living cell, wherein these are at least partly derived from at least one culture supernatant from at least one probiotic microorganism; and water, wherein the water is at least partly derived from at least one culture supernatant from at least one probiotic microorganism; and a base.

[0052] In a further aspect, the invention relates to a method of producing a composition, including the step of combining: water, wherein the water at least partly derived from at least one culture supernatant from at least one probiotic microorganism; and a base; to thereby produce the composition.

[0053] In a preferred embodiment, the present invention provides a method of producing a composition, including the step of combining: water, wherein the water at least partly derived from at least one culture supernatant from at least one probiotic microorganism; and supernatants, wherein the supernatant contains molecules and water at least partly derived from at least one culture from at least one probiotic microorganism; and supernatant molecules, wherein isolated and / or purified and / or extracted molecules are at least partly derived from at least one culture supernatant from at least one probiotic microorganism; and a base; to thereby produce a composition.

[0054] Suitably, the composition is that of the first aspect.

[0055] In a yet further aspect, the invention provides a composition produced according to the method of the second aspect.

[0056] Suitably, the composition of the first and third aspects is for use in:

[0057] (i) supplementation;

[0058] (ii) improving the health; and / or

[0059] (iii) the prevention and / or treatment of a disease, disorder or condition; of a subject.

[0060] In some embodiments, the disease, disorder or condition is selected from the group consisting of hypertension, heart disease, anxiety, obesity, a neurodegenerative disease, Alzheimer’s disease, a cancer, immunodeficiency, premature ageing, wrinkles, inflammation, gastrointestinal dysfunction, arthritis, osteoarthritis, allergies, molecular and gene dysregulations, gut microbiome disfunction, hormonal imbalance, vitamin depletion and any combination thereof.

[0061] In a fourth aspect, the invention resides in a method of supplementation in a subject, said method including the step of administering to said subject a therapeutically effective amount of the composition of the first and third aspects, to thereby supplement the subject.

[0062] In a fifth aspect, the invention relates to a method of improving the health of a subject, or maintaining the good health of a subject, said method including the step of administering to said subject a therapeutically effective amount of the composition of the first and third aspects, to thereby improve or maintain the health of the subject.

[0063] In some embodiments, improving the health of the subject comprises at least one of the following: reducing body weight, controlling blood sugar, increasing gut villi per area, increasing in average length of gut villi, promoting healthier intestinal mucosal barrier, increasing resistance to mechanical stress, preventing lean weight loss and / or to promoting weight increase during body growth, improving the efficiency of conversion of food into energy and muscle gain, preventing or reducing intra-abdominal fat accumulation, normalising blood biochemistry through probiotic ingestion, and / or improving gut biology, optionally despite consumption of gluten and fat-rich foods.

[0064] In some certain embodiments, the present invention may demonstrate the potential contribution of probiotics towards sustainable global protein increase, which is important as dietary protein is known to be crucial for optimal health (particularly during growth and aging).

[0065] In certain embodiments, probiotic cells may also be present aiming to enhance the effect of the supernatant and / or the secreted / shed molecules found in the supernatant, whereby the concentration of the cells in the composition or ingredient or supplement or medication or food additive or other applications thereof is 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x IO10, 1 x 1011, 5 x 105, 5 x 106, 5 x 107, 5 x 108, 5 x 109, 5 x IO10, 5 x 1011, or any other CFUs / recommended dose between 1 x 105and 5 x 1011.

[0066] Suitably, administration of the composition results in a substantial microbiota change. Preferably, the substantial microbiota change comprises an increase of at least one of the following microorganisms: Anaerococcus, Romboutsia, Lachnospiraceae, Lactobacillus o Bacillus, but it is not restricted to these microorganisms. Preferably, the substantial microbiota change comprises a decrease of at least one of the following microorganisms: Escherichia, Shigella, Clostridium, Salmonella, and other pathogens including coliforms and non-coliform microorganisms.

[0067] In a sixth aspect, the invention provides a method of treating and / or preventing a disease, disorder or condition in a subject, said method including the step of administering to said subject a therapeutically effective amount of the composition of the first and third aspects, to thereby treat and / or prevent the disease, disorder or condition of the subject.

[0068] Suitably, the disease, disorder or condition is selected from the group consisting of hypertension, heart disease, anxiety, obesity, a neurodegenerative disease, Alzheimer’s disease, a cancer, immunodeficiency, premature ageing, wrinkles, inflammation, gastrointestinal dysfunction, arthritis, osteoarthritis and any combination thereof.

[0069] In a seventh aspect, the invention provides a method of extending the shelf-life in a drink, food, beverage, cosmetic or other products containing the invention, said method including the step of adding an effective amount of the composition or the supernatant or a supernatant molecule of the first aspect, to thereby prevent and / or reduce and / or slow and / or decelerate and / or protect against spoilage.

[0070] Suitably, spoilage is caused by a group of biotic factors consisting of bacteria, fungi, virus, parasites, invertebrates, algae or protozoa and any combination thereof.

[0071] Suitably, spoilage is caused by a group of abiotic factors consisting of light, gases, temperature, surfaces, molecules, and any combination thereof.

[0072] With respect to the above aspects, the subject is suitably a human or other animal.

[0073] Throughout this specification, unless otherwise indicated, "comprise", "comprises" and “ comprising are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers, for example other additives, components, integers or steps. Conversely, the terms “consist”, “consists” and “consisting ' are used exclusively, such that a stated integer or group of integers are required or mandatory, and no other integers may be present.

[0074] The phrase “consisting essentially of indicates that a stated integer or group of integers are required or mandatory, but that other elements that do not interfere with or contribute to the activity or action of the stated integer or group of integers are optional.

[0075] As used in this specification the indefinite articles “a” and “an” may refer to one entity or a plurality of entities and are not to be read or understood as being limited to a single entity. For example, “a” subject includes one subject, one or more subjects or a plurality of subjects.

[0076] As used in this specification, the term “about” refers to a variation or tolerance in a stated amount, concentration, ratio or proportion, preferably defined as being no more than 10%, 5%, 2% or 1% above or below a stated amount, concentration, ratio or proportion. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0078] Figure 1 - Benefits promoted by the secretome of a probiotic fungus belonging to the Sclerodermataceae family, which is achieved through a composition containing supernatant, an additional 5% whey protein, and which is further enhanced by the presence of the living microbial cells that continuously produce supernatant molecules, as well as by the presence of spores that sensibilize the eukaryotic cells to better respond to the treatment: a) body weight of animals; b) body fat and blood sugar of animals;

[0079] Figure 2 - Independent histological analysis of the small intestine villi: a) untreated rats; b) treated with 5xl07probiotic fungus (Sclerodermataceae isolate TF18), magnification 40X.

[0080] Figure 3 - Weight gain promoted by the secretome of a probiotic lactic acid bacterium (Lactococcus lactis isolate QF16), which is further enhanced by the presence of the living cell continuously secreting molecules that constitute a supernatant: a) and b) low concentration group found to have increased weight by 6.5%, despite all animals receiving the same amount of feed; c) body weight was also found to increase faster among the low concentration treatment group.

[0081] DETAILED DESCRIPTION

[0082] The present inventors have created a composition, which includes molecules, molecular structures and water derived from culture supernatants used in the growth of probiotic microorganisms. Accordingly, the composition advantageously reduces water waste from the probiotic industry, whilst utilising culture supernatants that include beneficial molecules secreted by probiotic microorganisms grown therein. The compositions described herein are designed to, for example, be used for supplementation, to improve health and / or treat and / or prevent a range of diseases, disorders or conditions in a subject. Suitably, the composition is in the form of a dietary supplement, a fortified food product, a medicament and / or a stimulant.

[0083] In a first aspect, the invention resides in a composition comprising, consisting or consisting essentially of: water, wherein the water is at least partly derived from at least one culture supernatant from at least one probiotic microorganism; and a base.

[0084] The term “ culture supernatant” refers to the cell-free liquid component remaining when cells grown in a culture medium are separated therefrom by, for example, centrifugation, filtration, sedimentation, or any other means well known in the art.

[0085] Generally, when probiotic microorganisms, such as those described herein, are cultured in a medium, particular substances or molecules (e.g., proteins, polysaccharides, lipids) produced and contained within the probiotic microorganism are released or secreted therefrom. Accordingly, a culture supernatant can include a mixture of soluble components or metabolites found in the culture medium. It is envisaged that the culture supernatant can be taken from one or more growth phases or stages (e.g., the “lag”, the “log” (“logarithmic” or “exponential”), the “stationary” and the “death” (or “logarithmic decline”) phases) of a probiotic batch-cultivation process. In some embodiments, the composition includes a culture supernatant derived from an exponential growth phase of a probiotic batch-cultivation process.

[0086] When using the supernatant of probiotic cultures, the supernatant may be used as is or may be subjected to one or more purification steps before being added into the composition, such as to concentrate or isolate the active additives or metabolites therein.

[0087] As generally used herein, the term "probiotic" or "probiotic microorganism” refers to one or more living microorganisms that when administered in adequate amounts to an animal may confer a health benefit to said animal. This health benefit is typically the result of the probiotic beneficially modulating the animal’s gastrointestinal microbial balance or flora. Suitably, the probiotic microorganism is a bacterium or a fungus. Broadly, probiotic microorganisms may be of genera selected from the group consisting of Lactococcus, Sclerodermataceae, Lactobacillus, Bifidobacterium, Enterococcus, Streptococcus, Bacillus, Propionibacterium, Enterococcus, Streptococcus, Pediococcus, Clostridium, Aspergillus, Candida, Saccharomyces and Megasphaera, although without limitation thereto. In preferred embodiments, the probiotic microorganism is Lactococcus lactis isolate QF16 or Sclerodermataceae isolate TF18.

[0088] Lactococcus lactis isolate QF16 is a bacterial probiotic, that is a safe lactic acidproducing bacterium, traditionally used by the dairy industry. The isolate herein is innovative in containing natural antioxidant and metabolic genes.

[0089] Sclerodermataceae isolate TF18 is a fungal probiotic. The isolate is a natural filamentous fungus, with anti-cancer properties. The term “base” as used herein refers to a base composition, which is useful for preparing drink or food. By way of example, the base can be a fruit, vegetable or dairy base to which other ingredients, such as the culture supernatant and other additives, may be added to make up the composition.

[0090] Suitably, the base comprises a protein source, a carbohydrate source and / or a lipid source. In some embodiments, the base is or comprises: a protein source; a carbohydrate source; a lipid source; a protein source and a carbohydrate source; a protein source and a lipid source; a carbohydrate source and a lipid source; or a protein source, a carbohydrate source and a lipid source. It will be appreciated that two or more of the protein source, the carbohydrate source and the lipid source may be derived from a single source, such as those described herein. By way of example, a fruit juice or extract can be a source of protein, carbohydrates and lipid for the base. In alternative embodiments, each of the protein source, the carbohydrate source and the lipid source are derived from a difference source. Further to the above, the protein source, the carbohydrate source and / or the lipid source can be derived from multiple sources (e.g., two or more fruit juices, powders or extracts).

[0091] As used herein the term “protein” includes any amino acid polymer comprising D- or L-amino acids, natural or non-natural amino acids as known in the art. Proteins, peptides and / or amino acids may be in the form of extracts, hydrolysates, purified, chemical synthetic, enriched or partially-purified forms.

[0092] In some embodiments, the protein source may be obtained, derived, obtainable or derivable from natural sources such as plants and / or animals.

[0093] Animal sources include vertebrates such as mammals, reptiles, fish, and / or birds and invertebrates such as shellfish, crabs, lobsters and / or insects, although without limitation thereto. Non-limiting examples include whey protein concentrate, whey protein isolate, hydrolysed whey, casein, milk protein isolate, proteins from egg albumin, beef, lamb, pork, veal, venison, bison, goat, kangaroo, chicken, turkey, duck, emu, rabbit, fish, shellfish, crabs, lobster and / or crocodile, although without limitation thereto.

[0094] Plant sources include trees, vines, plant seeds, bark, roots, leaves, bulbs, tubers, nuts and / or fruit, although without limitation thereto. Plants may include grasses, cereals, legumes, seaweed, leafy vegetables, cruciform vegetables and fungi, although without limitation thereto. Non-limiting examples include soy protein, tofu, tempeh, pea protein, rice protein, bean proteins, corn protein, chickpea, faba bean, mungbean, chia protein, quinoa protein, spirulina protein, hemp seeds, lentils, nut proteins, heat germ protein, sorghum protein and proteins obtained, derived, obtainable or derivable from vegetables and / or fruit such as spinach, watercress, kale, broccoli, brussel sprouts, cauliflower, mushrooms, parsley, lettuce, zucchini, cucumber, capsicum, artichokes, cabbage, celery, eggplant, tomatoes, onion, beets, squash, pumpkin, potato, yams and / or sweet potato.

[0095] The term "carbohydrate" (or saccharides) encompasses monosaccharides, disaccharides, oligosaccharides and polysaccharides. In general, smaller (low molecular weight) carbohydrates, monosaccharides and disaccharides, are commonly referred to as sugars.

[0096] As used herein, the term “UpicT refers to a group of organic compounds that has lipophilic or amphiphilic properties, including, but not limited to, fats, fatty oils, essential oils, flavour oils, waxes, steroids, sterols, phospholipids, glycolipids, and fatty acids. The term “UpicT encompasses both naturally occurring and synthetically produced lipids.

[0097] It is envisaged that the lipid source can include, for example, any edible oil, butter or the like, including natural or synthesized oils or butters, which are suitable for human consumption. Exemplary lipid sources may include vegetable oils and butters, such as soybean, safflower, sunflower, sesame, peanut, com, olive, rice bran, canola, coconut oil, pomegranate seed oil, palm, cottonseed, rapeseed, carrot, evening primrose, borage, mustard, citrus seed, linseed, chia, avocado, hemp, sugarcane, macadamia, brazil nut, almond, beech nut, hazelnut, cashew, pecan, pine nut, mongongo, pistachio, walnut, pumpkin seed, grapefruit seed, melon seed, gourd seed, blackcurrant, evening primrose, cumin seed, amaranth oil, quinoa oil, apricot seed oil, apple seed, argan, babassu, ben, chestnut, chin nut, carob, cocoa, cocklebur, coriander seed, date seed, dika, grape seed, kapok, kenaf, lallemantia, mefura, marula, niger seed, nutmeg, papaya seed, perilla seed, persimmon seed, pequi, pili nut, poppyseed, pracaxi, prune kernel, ramtil, royle, sacha inchi, sapote, seje, shea, taramira, tes seed, thistle oil, tigemut, tabacco seed, tomato seed, wheat germ, colza, chaulmoogra, candlenut, neem, ojon, passionfruit, rose hip, sea buckthorn, viburnum, tonka bean, ucuhuba seed and meadowfoam. Additionally, mixtures of oils, butters and / or fractions thereof may be used to perform the present invention.

[0098] Suitably, one or more of the protein source, the carbohydrate source and the lipid source are derived or obtained from a non-animal source. Broadly, the non-animal-derived carbohydrate, protein and lipid sources may be obtainable or derivable, at least in part, from any non-animal source, such as microbe-derived (e.g., bacterial-derived, fungal- derived and algal-derived) sources and / or plant-derived sources, such as a plant extract. The terms “extract"", “component" and “derivative" refer to any product / s which may be derived from, for example, plant-based sources using a downstream processing technique or techniques (e.g., a series of techniques) known in the art, such as extraction and purification techniques.

[0099] In particular embodiments, one or more of the various components of the composition, such as the carbohydrate source, the protein source and / or the lipid source, are isolated or substantially purified. For the purposes of this invention, by “isolated" or “purified" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated or purified material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

[0100] By way of example, plant-derived or plant-based carbohydrate, protein and lipid sources may be obtainable or derivable from any plant or plant part. Plant-based sources include trees, vines, plant seeds, flowers, bark, roots, leaves, bulbs, tubers, nuts and / or fruit, although without limitation thereto. Plants may include fruits, grasses, cereals, legumes, seaweed, leafy vegetables, cruciform vegetables, mosses and fungi, although without limitation thereto. More particularly, the protein source, the carbohydrate source and / or the lipid source may be derived at least in part from: a plant extract, a vegetable extract, a root extract, a fruit extract, such as a fruit powder or a fruit juice; a flower extract, such as a flower powder; and / or a seed extract, such as a seed powder, a seed oil or a seed butter.

[0101] Accordingly, in various embodiments, the base is or comprises a fruit extract and / or a vegetable extract. The fruit and / or vegetable extracts may be included in the composition as a liquid, such as a juice, a liquid concentrate, a puree, a powder or in another modified form. The liquid from the fruit and / or vegetable may be filtered prior to being used in the composition. The fruit extract can include, for example, juice and powders from tomatoes, berries, citrus fruit, melons and / or tropical fruits. A single fruit juice or extract or fruit juice or extract blends may be used. The vegetable extract can include a number of different vegetable juices, powders etc. Exemplary fruit and vegetable extracts, such as a fruit or vegetable juice or powder, can be obtained or derived from berries of all types, currants, apricots, peaches, nectarines, plums, cherries, apples, pears, oranges, grapefruits, coconuts, lemons, limes, tangerines, mandarin, tangelo, bananas, pineapples, grapes, tomatoes, rhubarbs, prunes, figs, pomegranates, passion fruit, guava, kiwi, kumquat, mango, avocados, all types of melon, papaya, turnips, rutabagas, carrots, cabbage, cucumbers, squash, celery, radishes, Australian indigenous fruits / legumes / vegetables, bean sprouts, alfalfa sprouts, bamboo shoots, beans and / or seaweed. As can be appreciated, one or more fruits, one or more vegetables, and / or one or more fruits and vegetables, can be included in the composition to obtain the desired flavour thereof.

[0102] In specific embodiments, the protein source, the carbohydrate source and / or the lipid source are selected from the group consisting of cocoa powder, cacao powder, whey powder, milk powder, cacao butter, vegetable oils, coconut milk, lemon powder, honey powder, orange powder, berry powder, hibiscus powder, native Australian bush tucker (fruits and flowers) powder, tropical fruit and flower powder, and any combination thereof, whereby powder ingredients may be replaced by puree, mash, extracts, essences, liquors, liquids, juices and / or concentrates.

[0103] Suitably, the composition is a substantially vegetarian and / or vegan composition. To this end, the composition may be substantially free of animal-derived components.

[0104] As used herein, the term "vegetarian" refers to a product, composition or formulation, including but not limited to foods, drinks and supplements, which are not made from or with the aid of products derived from animals that have died, have been slaughtered, or animals that die as a result of being eaten. Such animals may include farmed, wild or domestic animals, including, but not limited to, livestock, poultry, game, fish, shellfish, Crustacea, amphibians, tunicates, echinoderms, molluscs and insects. In one example, a product can be vegetarian if it includes dairy products and eggs.

[0105] As used herein, the term “vegan” refers to a product including, but not limited to foods and supplements, which are not made from or with the aid of animals or animal products (including products from living animals).

[0106] In particular embodiments, the composition of the present aspect is in the form of a supplement, such as a protein supplement. As used herein, the term “supplement” refers to a supplement intended to supplement a diet of food and water, where the diet is sufficient to support life. A supplement may contain vitamins, minerals, herbs or other botanicals, amino acids, enzymes, organ tissues, glandular metabolites, or combinations thereof (e.g., a fortified food product).

[0107] In some embodiments, the composition is a single-serving shot, a single-serving unit or a multi-serving product. In other embodiments, the composition may be in a concentrated form, such as a syrup, cordial or powder, that requires the addition of, for example, additional water to a dilute form prior to ingestion by a subject.

[0108] As generally used herein, the stated concentration, proportion or mass of any particular component or constituent of the composition may be made with reference to the concentration, proportion or mass of the particular component or constituent used to produce the composition, or may be the concentration, proportion or mass of the particular component or constituent in the final product.

[0109] It is also noted that the stated concentrations, proportions or masses of each component or constituent may be readily varied by a person of skill in the art. It will also be appreciated that concentrations, proportions or masses expressed in the range “x-y” include all values and sub-ranges within the stated range.

[0110] The composition suitably includes the at least one culture supernatant from the at least one probiotic microorganism in a wt% concentration of about 0.1% to about 99% or any range therein such as, but not limited to, about 1% to about 75%, about 2% to about 30% or about 5% to about 20%. In particular embodiments, the at least one culture supernatant from the at least one probiotic microorganism is present at a wt% concentration of about 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5,

[0111] 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5,

[0112] 9.75, 10, 10.25, 10.5, 10.75, 11, 11.25, 11.5, 11.75, 12, 12.25, 12.5, 12.75, 13, 13.25, 13.5,

[0113] 13.75, 14, 14.25, 14.5, 14.75, 15, 15.25, 15.5, 15.75, 16, 16.25, 16.5, 16.75, 17, 17.25, 17.5, 17.75, 18, 18.25, 18.5, 18.75, 19, 19.25, 19.5, 19.75, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % and any range therein. In particularly preferred embodiments, the composition comprises from about 1 wt% to about 50 wt% of the at least one culture supernatant from the at least one probiotic microorganism.

[0114] The composition suitably includes the base in a wt% concentration of about 1% to about 99% or any range therein such as, but not limited to, about 1% to about 75%, about 2% to about 50% or about 5% to about 40%. In particular embodiments, the base is present at a wt% concentration of about 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 10.25, 10.5, 10.75, 11, 11.25, 11.5, 11.75, 12, 12.25, 12.5, 12.75, 13, 13.25, 13.5, 13.75, 14, 14.25, 14.5, 14.75, 15, 15.25, 15.5, 15.75, 16, 16.25, 16.5, 16.75, 17, 17.25, 17.5, 17.75, 18, 18.25, 18.5, 18.75, 19, 19.25, 19.5, 19.75, 20, 21, 22,

[0115] 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,

[0116] 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,

[0117] 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

[0118] 95, 96, 97, 98, 99 % and any range therein. In particularly preferred embodiments, the composition comprises from about 10 wt% to about 90 wt% of the at least one culture supernatant from the at least one probiotic microorganism.

[0119] Suitably, the composition comprises from about 0.1 wt% to about 99 wt% (e.g., about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

[0120] 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

[0121] 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,

[0122] 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,

[0123] 96, 97, 98, 99 % and any range therein) of the protein source, such as those hereinbefore described. In particularly preferred embodiments, the composition comprises from about 1 wt% to about 20 wt% of the protein source.

[0124] Suitably, the composition comprises from about 0.1 wt% to about 99 wt% (e.g., about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

[0125] 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

[0126] 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,

[0127] 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,

[0128] 96, 97, 98, 99 % and any range therein) of the carbohydrate source, such as those hereinbefore described. In particularly preferred embodiments, the composition comprises from about 1 wt% to about 20 wt% of the carbohydrate source.

[0129] Suitably, the composition comprises from about 0.1 wt% to about 99 wt% (e.g., about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

[0130] 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

[0131] 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,

[0132] 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,

[0133] 96, 97, 98, 99 % and any range therein) of the lipid source, such as those hereinbefore described. In particularly preferred embodiments, the composition comprises from about 1 wt% to about 10 wt% of the lipid source.

[0134] In some embodiments, the base further comprises a sweetening agent.

[0135] As generally used herein, the term " sweetening agent" refers to natural or artificial compounds used to increase the sweetness of the present composition. It will be appreciated that sweetening agents include carbohydrate sweetening agents, (i.e., sugars and other carbohydrate sweetening agents) and non-carbohydrate sweetening agents. Accordingly, the sweetening agent may also be considered to be a carbohydrate source and vice versa. As used herein, the term "sugar" refers to sucrose and the constituents of sucrose (e.g., glucose and / or fructose, sugar syrup, malt syrup, maple syrup, starch syrup, glucose syrup, high-fructose syrups such as high-fructose com syrup, honey, molasses) and other carbohydrates that can be used as sweetening agents or a source of these. The term "other carbohydrate sweetening agents" refers to, for example, sugar alcohols, such as xylitol, maltitol, lactitol, glycerol and sorbitol. Suitable examples of the noncarbohydrate sweetening agents include e.g. stevia, aspartame (phenylalanine), acesulfame potassium (Ace-K), saccharin, cyclamates and sucralose. Accordingly, the sweetener may include a low calorie sweetener, a natural sweetener, a non-nutritive sweetener and / or an artificial sweetener. Additionally, the sweetener may be naturally or synthetically derived.

[0136] Suitably, the sweetening agent or combination of sweetening agents are present at a concentration of about 0.01 to about 30 wt% (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 wt% and any range therein), more preferably about 0.05 to about 20 wt% or even more preferably about 0.1 to about 15 wt%.

[0137] In some embodiments, the composition further comprises one or more of a pH modifier, a preservative, a solvent, an emulsion stabilizer, a colouring agent, a flavouring agent, an amino acid, a vitamin, prebiotic fibre and a mineral. In some embodiments, the composition is substantially free of artificial sweetening agents, artificial flavouring agents and / or artificial colouring agents.

[0138] Suitably, the amino acid can be any known in the art, inclusive of essential and non-essential amino acids, as well as derivatives or variants thereof, such as amino acid salts. Nonlimiting examples include leucine, isoleucine, valine, lysine, glutamine, threonine, phenylalanine, methionine, tryptophan, histidine, taurine, arginine, serine, proline, glycine, alanine, ornithine, citrulline, urocanic acid, asparagine and tyrosine.

[0139] With respect to the present aspect, the composition may include one or more amino acids in a wt% concentration of about 0.1% to about 20% (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5,

[0140] 9.75, 10, 10.25, 10.5, 10.75, 11, 11.25, 11.5, 11.75, 12, 12.25, 12.5, 12.75, 13, 13.25, 13.5,

[0141] 13.75, 14, 14.25, 14.5, 14.75, 15, 15.25, 15.5, 15.75, 16, 16.25, 16.5, 16.75, 17, 17.25,

[0142] 17.5, 17.75, 18, 18.25, 18.5, 18.75, 19, 19.25, 19.5, 19.75, 20) or any range therein, such as, but not limited to, about 1% to about 15% or about 2% to about 5%.

[0143] It will be appreciated that the colouring agent may be any as are known in the art. To this end, a wide range of food grade colouring agents are used by the food industry in order to enhance the optical appearance of food, dietary supplements and the like. Of the known food grade colouring agents, only a few are natural, such as betanoin or chlorophyll, for instance. Most food grade colouring agents are either synthetic analogues of naturally occurring substances - so-called nature identical substances - or are fully artificial.

[0144] Further to the above, the composition of the invention can include one or more colouring agents, such as those hereinbefore described, in a wt% concentration of about 0.1% to about 20% (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 10.25, 10.5, 10.75, 11, 11.25,

[0145] 11.5, 11.75, 12, 12.25, 12.5, 12.75, 13, 13.25, 13.5, 13.75, 14, 14.25, 14.5, 14.75, 15, 15.25, 15.5, 15.75, 16, 16.25, 16.5, 16.75, 17, 17.25, 17.5, 17.75, 18, 18.25, 18.5, 18.75, 19, 19.25, 19.5, 19.75, 20) or any range therein such as, but not limited to, about 1% to about 15% or about 2% to about 12%.

[0146] The flavouring agents of the invention may be any natural or non-natural substance which adds or enhances flavour. Flavouring agents are typically comprised of a flavoured substance(s) and complexes manufactured or extracted from nature in liquid or powdered form to impart a particular flavour into a product.

[0147] Referring to the present aspect, the composition may include one or more flavouring agents, such as those hereinbefore described, in a wt% concentration of about 0.1% to about 20% (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 10.25, 10.5, 10.75, 11, 11.25,

[0148] 11.5, 11.75, 12, 12.25, 12.5, 12.75, 13, 13.25, 13.5, 13.75, 14, 14.25, 14.5, 14.75, 15, 15.25, 15.5, 15.75, 16, 16.25, 16.5, 16.75, 17, 17.25, 17.5, 17.75, 18, 18.25, 18.5, 18.75, 19, 19.25, 19.5, 19.75, 20) or any range therein such as, but not limited to, about 1% to about 15% or about 2% to about 10%. Suitably, the composition of the present aspect further comprises one or more vitamins and / or minerals, such as electrolytes. Exemplary vitamins include vitamin A (e.g., vitamin Ai retinol, axerophthol, a-carotene, P-carotene, y-carotene), B vitamins (e.g., Bi vitamins including: thiamin, aneurin, thiamine, pyrophosphate, cocarboxylase; B2 vitamins including riboflavin, vitamin G, lactoflavin, hepatoflavin, ovoflavin, verdoflavin, riboflavin mononucleotide, FMN, riboflavin dinucleotide, FAD; Vitamin B3 (niacin), Vitamin Be (pyridoxine, pyridoxal-5-phosphate), Vitamin B7 (biotin), Vitamin B12 (cobalamin, methylcobalamin)), Vitamin C (ascorbic acid, antiscorbutic vitamin, dehydroascorbic acid), D vitamins (antirachitic vitamin, vitamin D2, D3, cholecalciferol, etc), Vitamin E, Vitamin K, and the like. Nonlimiting examples of minerals include sodium, selenium, zinc, magnesium, calcium, iron, manganese, copper, chromium, phosphorous, iodine, potassium and molybdenum.

[0149] In some embodiments, the composition further comprises a pH modifier. The pH modifier may be any agent, substance or molecule that facilitates obtaining and / or maintaining an optimal pH for the composition. The pH modifier may be a buffering agent, acid or base that facilitates achieving an acidic pH, an alkaline pH or neutral pH (i.e., about pH 7). Typically, the pH modifier is an acid, such as an organic acid. Organic acids may be any food acid, such as a carboxylic acid, that is suitable for human consumption and includes salts of said organic acids. Non-limiting examples include tartaric acid, citric acid, lactic acid, benzoic acid, acetic acid, malic acid, ascorbic acid, succinic acid, fumaric acid, oxalic acid and their salts (e.g tartrates, citrates, lactates etc). Preferred organic acids are citric acid, malic acid and ascorbic acid.

[0150] Suitably, the pH modifiers are present at a concentration of about 1-10% (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 wt% or any range therein) by weight in total, or preferably about 2-7% by weight in total.

[0151] The composition can further contain one or more emulsion stabilizers (co- emulsifiers), which can be used to prevent phase separation and stabilize the composition. Exemplary emulsion stabilizers include gums, such as xanthan gum, guar gum, sodium alginate, gum acacia, ester gums and sugar beet pectin, lecithins, such as soy lecithin, and modified food starches.

[0152] The preservative may be any as are well known in the art. Exemplary preservatives include benzoates such as sodium benzoate, potassium benzoate, calcium benzoate, sodium sorbate, potassium sorbate, calcium sorbate, sodium citrate, potassium citrate, polyphophates such as sodium hexametaphosphate, antioxidants such as ascorbic acid, ethylenediaminetetraacetic acid, beta hydroxy acid, butylhydroxytoluene, tertiary butylhydroquinone, dehydroacetic acid, dimethyldicarbonate, ethoxyquin and heptylparaben.

[0153] In various embodiments, the composition is substantially gluten free. In this regard, the composition can be substantially free of gluten-containing products, such as gluten-containing flours and starches (e.g., has no more than about 0.5%, 0.2%, 0.1% or 0.05% gluten by weight thereof).

[0154] In particular embodiments, the composition is substantially lactose free. To this end, the composition can be substantially free of milk or dairy-based or derived products (e.g., has no more than about 0.5%, 0.2%, 0.1% or 0.05% milk or dairy -based or derived lactose by weight thereof).

[0155] In one particular embodiment of the present aspect, the composition is a protein supplement comprising:

[0156] (i) at least one culture supernatant from at least one probiotic microorganism;

[0157] (ii) a protein source, a carbohydrate source and a lipid source comprising a seed extract and / or a fruit extract;

[0158] (iii) a sweetening agent; and

[0159] (i) optionally a pH modifier, a preservative, a solvent, an emulsion stabilizer, a colouring agent, a flavouring agent, an amino acid, prebiotic fibre, a vitamin and a mineral.

[0160] In another embodiment of the present aspect, the composition comprises:

[0161] (i) at least one culture supernatant from at least one probiotic microorganism;

[0162] (ii) a protein source, a carbohydrate source and a lipid source comprising a fruit extract and / or a flower extract;

[0163] (iii) a sweetening agent; and

[0164] (i) optionally a pH modifier, a preservative, a solvent, an emulsion stabilizer, a colouring agent, a flavouring agent, an amino acid, a vitamin and a mineral.

[0165] Given the included components, it will be appreciated by the skilled person that the composition may be for use in:

[0166] (i) supplementation, such as protein supplementation;

[0167] (ii) improving health, such as gastrointestinal health; and / or

[0168] (iii) the prevention and / or treatment of a disease, disorder or condition; of a subject. In some embodiments, the disease, disorder or condition is selected from the group consisting of hypertension, heart disease, anxiety, obesity, a neurodegenerative disease, Alzheimer’s disease, a cancer, immunodeficiency, premature ageing, wrinkles, inflammation, gastrointestinal dysfunction, arthritis, osteoarthritis, allergies, molecular and gene dysregulations, gut microbiome disfunction, hormonal imbalance, vitamin depletion and any combination thereof.

[0169] With respect to improving gastrointestinal health, administration of the composition described herein may modulate one or more species or genera of microbial flora in at least a portion of a gastrointestinal tract of a subject. In this regard, the composition may be considered a modbiotic.

[0170] In a further aspect, the invention relates to a method of producing a composition, including the step of combining: water, wherein the water at least partly derived from at least one culture supernatant from at least one probiotic microorganism; and a base; to thereby produce the composition.

[0171] Suitably, the composition is that of the first aspect.

[0172] In some embodiments, the method further includes combining one or more of a pH modifier, a preservative, a solvent, an emulsion stabilizer, a colouring agent, a flavouring agent, an amino acid, prebiotic fibre, a vitamin and a mineral.

[0173] Suitably, the composition is according to that described herein.

[0174] In another aspect, the invention provides a composition produced according to the aforementioned method.

[0175] It is envisaged that the previously described composition may be suitable for use in providing nutritional supplementation, such as protein supplementation, to a subject in need thereof.

[0176] Accordingly, in yet a further aspect, the invention resides in a method of supplementation in a subject, said method including the step of administering to said subject a therapeutically effective amount of the composition described herein, to thereby supplement the subject.

[0177] By “administration” or “administering” is meant the introduction of a composition (e.g., a composition comprising, consisting or consisting essentially of water, wherein the water at least partly derived from a culture supernatant from a probiotic microorganism; and a base) into a subject by a chosen route, and in particular by the oral route.

[0178] The term “therapeutically effective amount” describes a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of the composition hereinbefore described to: (a) provide adequate supplementation; (b) promote health; and / or (b) reduce, alleviate and / or prevent a disease, disorder or condition in a subject. In some embodiments, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of such a disease, disorder or condition or achieve at least partly a recommended daily intake of, for example, protein, an amino acid, a vitamin or a mineral. In other embodiments, a “therapeutically effective amount” is an amount sufficient to achieve a desired biological effect.

[0179] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of the composition useful for, for example, reducing, alleviating and / or preventing a disease, disorder or condition will be dependent on the subject being treated, the type and severity of any associated disease, disorder and / or condition, and the manner of administration of the therapeutic composition.

[0180] In one embodiment, a given dosage of the composition, such as orally, is applied as a single application or a plurality of applications over a given time period (e.g., for as long as the subject requires treatment), where the dosing schedule is administered over a given time period, examples of which include hourly, daily, weekly, biweekly or monthly dosing schedules.

[0181] The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically / therapeutically-effective. The dose administered to a subject, in the context of the present invention, should be sufficient to effect a beneficial response (e.g., a reduction or amelioration in symptoms of a disease, disorder or condition) in a subject over an appropriate period of time. The quantity of the composition to be administered may depend on the subject to be treated, inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of a practitioner of ordinary skill in the art.

[0182] In particular embodiments, the present method is designed to supplement the nutritional or protein intake of subjects engaged in bodybuilding and protein supplementation, meal replacement, exercise recovery and / or mass gaining. In other embodiments, the present method is designed to supplement the nutritional or protein intake of subjects suffering from age-related and / or disease-related muscle loss, such as sarcopenia and muscle loss associated with rheumatoid arthritis, diabetes, hormonal imbalances, peripheral arterial disease, COPD, congestive heart failure, advanced kidney disease, cirrhosis, cancer, and HIV.

[0183] In a related aspect, the invention relates to a method of improving the health of a subject, said method including the step of administering to said subject a therapeutically effective amount of the composition described herein, to thereby improve the health of the subject.

[0184] In particular embodiments, the present method is designed to improve the gastrointestinal health of said subject.

[0185] In another aspect, the invention provides a method of treating and / or preventing a disease, disorder or condition in a subject, said method including the step of administering to said subject a therapeutically effective amount of the composition described herein, to thereby treat and / or prevent the disease, disorder or condition of the subject.

[0186] In another aspect, the invention provides a method of extending the shelf-life of a drink, beverage, food or a cosmetic, said method including the step of adding to said product an effective amount of the composition or ingredient described herein, to thereby prevent spoilage.

[0187] In particular embodiments, the present method is designed to ensure that the refrigerated shelf-life of a said product is at least 2.5 months, and to ensure that the roomtemperature shelf-life of a said product is at least 1 month.

[0188] As used herein, “treating', “treat' or “treatment' refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of a disease, disorder or condition after said disease, disorder or condition and / or its symptoms have at least started to develop. As used herein, “preventing', “prevent" or “prevention" refers to therapeutic intervention, course of action or protocol initiated prior to the onset of a disease, disorder or condition and / or a symptom thereof so as to prevent, inhibit or delay or development or progression of said disease, disorder or condition or the symptom. In this regard, a “prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease, disorder or condition or exhibits only early signs for the purpose of decreasing the risk of developing such a disease, disorder or condition.

[0189] Suitably, the disease, disorder or condition is selected from the group consisting of hypertension, heart disease, anxiety, obesity, a neurodegenerative disease, Alzheimer’s disease, a cancer, immunodeficiency, premature ageing, wrinkles, inflammation, gastrointestinal dysfunction, arthritis, osteoarthritis and any combination thereof.

[0190] The term “subject”, as used herein, includes both human and veterinary subjects. For example, administration to a subject can include administration to a human subject or a veterinary subject. Preferably, the subject is a human. However, therapeutic uses according to the invention may also be applicable to mammals such as domestic and companion animals, performance animals such as horses, livestock, and laboratory animals.

[0191] All patent and scientific literature referred to herein is incorporated herein by reference.

[0192] So that the present invention may be more readily understood and put into practical effect, the skilled person is referred to the following non-limiting examples.

[0193] EXAMPLES

[0194] The present invention will now be described with reference to the following examples which should be considered in all respects as illustrative and non-restrictive.

[0195] EXAMPLE 1

[0196] Example 1 provides an embodiment of the hydrating drink composition as a dietary protein and dietary fibre and vitamin supplement, which also contains a high antioxidant content due to the presence of carboxylic acids and low-concentration biogenic amines and amides.

[0197] Embodiment 1 (drink version: Cocoa Super Protein)

[0198] Ingredients and weight %

[0199] Supernatant of probiotic bacterial cultures - 10%

[0200] Cocoa powder - 1.5%

[0201] Cacao powder - 1.5%

[0202] Cacao butter - 0.5%

[0203] Coconut milk - 2%

[0204] Lemon powder - 0.2%

[0205] Manuka honey powder - 20% Glycerol - 1%

[0206] Stevia powder - 0.015%

[0207] Soy lecithin - 0.3%

[0208] Water - 62.985%

[0209] Preparation and production methods

[0210] The below methods were undertaken using aseptic techniques to help prevent and control contamination.

[0211] 1. Powder ingredients are weight and liquid ingredients are measured;

[0212] 2. The powder and liquid ingredients are dispensed into a container;

[0213] 3. Filtered water is added until it covers the powder and liquid ingredients;

[0214] 4. Stirring is undertaken at 30°C to ensure homogenisation of the mixture and solubilisation of all components;

[0215] 5. Supernatant of probiotic bacterial cultures are filtered using a 0.22 pm filter, sonicated using ultra-sound, or pasteurised as required, and then added to the base composition;

[0216] 6. Filtered water is added until the final intended volume is reached;

[0217] 7. The final liquid is dispensed into bottles;

[0218] 8. The filled bottles are packaged, palletised and cold-stored at 4°C;

[0219] 9. Bottles are randomly selected for quality-control;

[0220] 10. Satisfactory batches presenting CFU / g less than 10E2 for coliforms and non- coliforms are then shipped to commercial destinations.

[0221] EXAMPLE 2

[0222] Example 2 provides a further embodiment of the hydrating drink composition as a dietary fibre and vitamin supplement which also contains a high antioxidant content due to the presence of carboxylic acids and low-concentration biogenic amines and amides.

[0223] Embodiment 2 (drink version: Australian Berries)

[0224] Ingredients and weight %

[0225] Supernatant of probiotic bacterial cultures - 7%

[0226] Orange powder - 0.5% Mixed berries powder - 3%

[0227] Glycerol - 2%

[0228] Hibiscus powder - 0.5%

[0229] Lemon powder - 0.3%

[0230] Manuka honey - 10%

[0231] Xanthan gum - 0.01%

[0232] Water - 76.69%

[0233] Preparation and production methods

[0234] Are the same as in Example 1.

[0235] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms in particular features of any one of the various described examples may be provided in any combination in any of the other described examples. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows.

[0236] EXAMPLE 3

[0237] Example 3 shows preliminary experimental data as to the beneficial aspects of the probiotics of the present invention.

[0238] Probiotics

[0239] Lactococcus lactis isolate QF 16 and Sclerodermataceae isolate TF 18 are approved by DAF-QLD (SA 2020 / 08 / 750) for animal scientific use.

[0240] Stage 1

[0241] A composition comprising a mixture of dog pellets (comprising fat and carbohydrates), processed meats, food scraps, tap water, 5% protein supplement and a probiotic mixture containing species la and lb [species la and lb being Lactococcus lactis isolate QF16 and Sclerodermataceae isolate TF18] was administered to 10-y ear-old dogs, over a period of 30 days.

[0242] Increased body mobility and activity was reported alongside the absence of pain and / or toxicity.

[0243] Stage 2

[0244] 4-week-old laboratory rats were administered a composition over a period of 30 days. The composition was administered orally every second day for the first two weeks, followed by weekly oral administration for the final two weeks.

[0245] The composition comprised dog pellets (comprising fat and carbohydrates), organic fruits, tap water, 5% protein supplement, and a probiotic. The probiotic in the first group was a commercial probiotic [Blackmores PAW DigestiCare], The probiotic in the second group was fungal cells from the Sclerodermataceae isolate TF 18 at a concentration of 5 x 107. The probiotic in the third group was fungal cells from the Sclerodermataceae isolate TF18 at a concentration of 5 x 108. The control did not comprise a probiotic.

[0246] The results showed that animals receiving 5xl07cells presented body weight similar to untreated controls (Figure 1 A), however, the animals were leaner and showed better blood sugar level control (Figure IB). It was particularly surprising and unexpected that the Sclerodermataceae isolate TF18 produced a significantly improved result over the commercial probiotic.

[0247] Stage 3

[0248] Gut samples were collected from the above rat models after the 30-day administration period was completed. These samples were then analysed at a pathology laboratory. Gut histology was conducted following established protocols and the results show that the Sclerodermataceae isolate TF18 as compared to the untreated control group (Figure 2) exhibited:

[0249] • 83% increase in gut villi per area;

[0250] • 62% increase in average length of gut villi;

[0251] • healthier intestinal mucosal barrier, which is crucial for the precise translocation of water, nutrients and molecules between cells (e.g. gut-brain-endocrine responses); and increased resistance to mechanical stress.

[0252] Stage 4

[0253] Chickens were administered a composition over a period of 43 days in industry pens. The composition was administered orally on a weekly basis.

[0254] The composition comprised a whey protein supplement and a probiotic. The probiotic in the first group was the bacterial probiotic Lactococcus lactis QF16 at a concentration of 1 x 108or 8 x 107CFU / ml. The probiotic in the second group was the same bacterial probiotic at a concentration of 1 x 109CFU / ml. The control did not comprise a probiotic, and some controls comprise the medium only mixture without probiotic cells or their supernatants, resulting in the same outcome as untreated controls (data not shown).

[0255] The results showed that animals receiving 1 x 108or 1 x 107probiotic presented a significant weight gain, despite eating the same amount as untreated controls (Figure 3 A and B). Unexpectedly, the lower concentration bacterial probiotic combined with protein supplementation, was found to increase weight faster than the control of higher concentration of bacterial probiotic (Figure 3C).

[0256] Stage 5

[0257] Gut samples were collected from the above chicken models after the 43-day administration period was completed. These samples were then analysed at a pathology laboratory. Gut histology was conducted following established protocols and the results show that the group treated with probiotic la as compared to the untreated control group showed no signs of toxicity, tissue damage or cellular pathologies were observed among groups (Table 1).

[0258] Stage 6

[0259] The manure was collected from the above chicken models were also sent for external microbiome sequencing after 43 days, and the results were analysed following established bioinformatic protocols.

[0260] It was found that the gut tract of chickens treated with low concentration Lactococcus lactis QF16 (1 x 108or 8 x 107) had a substantial microbiota change in comparison to untreated controls. The following microorganisms were found in the gut tract of the treated chickens:

[0261] Anaerococcus (2.24-fold increase): this genus encompasses bacteria known to break down sugars, while producing natural butyrate. Herein, genus increase was found to correlate well with a better use of nutrition, which improved poultry body metabolism.

[0262] Romboutsia (4.39-fold increase): this genus encompasses bacteria known to be associated with healthy guts, which has been established by its presence in healthy humans. The maintenance of Rombutsia population plays a role in reducing / preventing gut alterations, and in fact this genus is common in the gut of poultry and the microflora of egg shells due to its involvement in energy metabolism and hormonal regulation. Herein the genus was induced by the treatment, and it can be correlated with accelerated growth and increased body weight.

[0263] Lachnospiraceae (10% population increase): this genus encompasses bacteria known to improve food conversion rate, whereby less food is needed to promote more gain. Microbes within this genus are highly abundant in animal trials with positive Feed Conversion Rate (FCR) results, as the bacteria herein are well documented regarding their ability to capture energy from food, produce acetate as an energy donor for muscle growth and fat bum, and also release natural propionates and butyrates that regulate FFAR3 gene expression to modulate fat deposition and gluconeogenesis. Lachnospiraceae also protects individuals through short-chain fatty acid production, and genus induction herein correlates with lean growth and possibly better meat quality.

[0264] Lactobacillus (2.21-fold increase): this genus encompasses bacteria known to increase cell proliferation and serum testosterone levels in male mice. In fact, L. reuteri population was increased by 88% herein. In other Lactobacillus studies, treated mice were also recognised as younger than untreated siblings based on physical assessments, suggesting a potential role for microbe therapy in public health, through positive impact on hormonal health and reproductive fitness (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3879365 / ). In our poultry trials, this genus was induced by the IxlO8or 8 x 107treatment and correlated with lean growth and active metabolism.

[0265] Bacillus (2.30-fold increase): this genus encompasses bacteria known to form spores, which are notorious for promoting probiotic stimulation of the gut and immune responses. Herein, genus inductions correlates with gut structural health and increased metabolic activity.

[0266] Escherichia and Shigella (3.84-fold reduction): these are known pathogens of humans and animals, and their significant reduction correlates with the increase of beneficial gut bacteria.

[0267] Conclusions

[0268] The study herein recommends the use of probiotics as a method boosting protein accumulation endogenously, by suggesting that when the human body is exposed to the right set of probiotics it can better utilise nutrition, incorporate more protein into muscles, and potentially help deliver global sustainability through the improved metabolism of foods.

[0269] The technologies developed herein are also rapidly scalable and are able to reduce the pressure on farming systems, ultimately creating a positive environmental impact by reducing greenhouse emissions and water consumption.

[0270]

Claims

CLAIMS1. A composition comprising: secreted molecules and shed molecules and / or probiotic cell structures that do not constitute the living cell, wherein these are at least partly derived from at least one culture supernatant from a cellular organism; and water, wherein the water is at least partly derived from at least one culture supernatant from at least one cellular organism; and a base.

2. The composition of Claim 1, wherein the cellular organism from which the culture supernatant is derived is a probiotic microorganism3. The composition of Claim 1 or 2, wherein the base comprises a protein source, a carbohydrate source, a lipid source and / or a sweetening agent.

4. The composition of Claim 3, wherein the protein source, the carbohydrate source and / or the lipid source are derived from one or more non-animal-sources.

5. The composition of Claim 4, wherein the protein source, the carbohydrate source and / or the lipid source are derived at least in part from a plant.

6. The composition of any one of Claims 3 to 5, wherein the protein source, the carbohydrate source and / or the lipid source are derived at least in part from a plant extract, a fruit extract, such as a fruit powder and a fruit juice, a flower extract, such as a flower powder, and / or a seed extract, such as a seed powder, seed oil and a seed butter.

7. The composition of any one of the preceding claims, wherein the composition is a substantially vegetarian and / or vegan composition.

8. The composition of any one of the preceding claims, wherein the composition comprises from about 0.1 wt% to about 99 wt% of the at least one culture supernatant from the at least one probiotic microorganism.

9. The composition of any one of the preceding claims, wherein the composition comprises from about 0.1 wt% to about 99 wt% of the base.

10. The composition of any one of Claims 2 to 8, wherein the composition comprises from about 0.1 wt% to about 30 wt% of the sweetening agent.

11. The composition of any one of the preceding claims, further comprising one or more of a pH modifier, a preservative, a solvent, an emulsion stabilizer, a colouring agent, a flavouring agent, prebiotic fibre, a vitamin and a mineral.

12. The composition of any one of the preceding claims, wherein the composition is in the form of a supplement.

13. The composition of Claim 12, wherein the composition is a protein supplement.

14. A method of producing a composition, including the step of combining: water, wherein the water at least partly derived from at least one culture supernatant from at least one cellular organism; and a base; to thereby produce the composition.

15. The method of Claim 14, wherein the cellular organism from which the culture supernatant is derived is a probiotic microorganism.

16. The method of Claims 14 or 15, wherein the composition is that according to any one of Claims 1 to 13.

17. A composition produced according to the method of Claim 14 to Claim 16.

18. A composition according to the any one of Claims 1 to 13 and 17, for use in:(i) supplementation;(ii) improving and / or maintaining health; and / or(iii) the prevention and / or treatment of a disease, disorder or condition; of a subject.

19. A method of supplementation in a subject, said method including the step of administering to said subject a therapeutically effective amount of the composition according to any one of Claims 1 to 13 and 17, to thereby supplement the subject.

20. A method of improving health in a subject, said method including the step of administering to said subject a therapeutically effective amount of the composition according to any one of Claims 1 to 13 and 17, to thereby improve the health of the subject.

21. The method of Claim 20, wherein improving the health of the subject comprises at least one of the following: reducing body weight, controlling blood sugar, increasing gut villi per area, increase in average length of gut villi, healthier intestinal mucosal barrier and / or increased resistance to mechanical stress.

22. The method of Claim 20 or 21, wherein the concentration of the composition is 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010, 1 x 1011, 5 x 105, 5 x 106, 5 x 107, 5 x 108, 5 x 109, 5 x 1010, 5 x 1011, or any other CFUs / recommended dose between 1 x 105and 5 x 1011.

23. The method of Claim 20 or 21, wherein administration of the composition results in a substantial microbiota change.

24. The method of Claim 23, wherein the substantial microbiota change comprises an increase of at least one of the following microorganisms: Anaerococcus, Romboutsia, Lachnospiraceae, Lactobacillus or Bacillus.

25. The method of Claim 233, wherein the substantial microbiota change comprises a decrease of at least one of the following microorganisms: Escherichia or Shigella or other Gram-positive or Gram-negative bacteria, or fungi or parasites or protozoa.

26. A method of treating and / or preventing a disease, disorder or condition in a subject, said method including the step of administering to said subject a therapeutically effective amount of the composition according to any one of Claims 1 to 13 and 17, to thereby treat and / or prevent the disease, disorder or condition of the subject.

27. The use of Claim 16 or the method of Claim 24, wherein the disease, disorder or condition is selected from the group consisting of hypertension, heart disease, anxiety, obesity, a neurodegenerative disease, Alzheimer’s disease, a cancer, immunodeficiency, premature ageing, wrinkles, inflammation, gastrointestinal dysfunction, arthritis, osteoarthritis, allergies, molecular and gene dysregulations, gut microbiome disfunction, hormonal imbalance, vitamin depletion and any combination thereof.

28. The method or use according to any one of Claims 16 to 25, wherein the subject is a human or animal.

29. The composition of Claim 1, wherein use of the composition or supernatants or supernatant molecules or shed molecules or shed cellular structures results in a cosmetic.

30. The composition of Claim 1, wherein use of the composition or supernatants or supernatant molecules or shed molecules or shed cellular structures results in shelf-life extension of an item stored under refrigeration and / or room temperature.

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