Stimulation of the gut microbiota to produce vitamins

Abstract

The invention relates to the non-therapeutic use of one or more lipids to stimulate gut microbiota to produce certain vitamins. The invention also relates to nutraceutical products comprising one or more lipids for stimulating the gut microbiota to produce certain vitamins. Lipids of particular interest are lauric acid (C12:0), oleic acid (C18:1), linoleic acid (C18:2). Vitamins of particular interest are riboflavin and pyridoxamine.

Description

[0001] STIMULATION OF THE GUT MICROBIOTA TO PRODUCE VITAMINS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the non-therapeutic use of one or more lipids to stimulate gut microbiota to produce certain vitamins. The invention also relates to nutraceutical products comprising one or more lipids for stimulating the gut microbiota to produce certain vitamins.

[0004] BACKGROUND OF THE INVENTION

[0005] Gut microbiota (also known as the gut microbiome or gut flora) are the microorganisms that live in the digestive tracts of animals. The gut microbiota includes bacteria, fungi, viruses and archaea. The composition of the gut microbiome varies between different regions of the digestive tract. In humans, the majority of organisms in the gut microbiome are found in the colon. It is believed that there are between three hundred and one thousand different species of microorganisms living in a typical human colon with the majority of these species being bacterial species. It is also believed that the vast majority of bacteria in the colon come from around 30 to 40 different bacterial species.

[0006] The gut microbiome plays an important and complex role in mammalian health and wellbeing. One role is in the direct inhibition of pathogens by competing with the pathogens for space and nutrients within the digestive tract. The gut microbiome also plays a key role in development of the host defence and immune system such as by promoting the development of gut tissue that functions as a barrier to pathogenic microorganisms and it plays a critical role in training and development of major components of the host’s immune system.

[0007] In addition, the gut microbiota plays a role in metabolism. Certain carbohydrates that cannot be broken down by human digestive enzymes fermented by the microbiome. For example, certain starches, fiber, sugars and oligosaccharides are fermented into short chain fatty acids. These can then be locally used as energy source for the enterocytes or absorbed by the host of the microbiome into the systemic circulation where they are associated with various health benefits. The gut microbiome is also believed to produce a variety of other metabolites such as amino acids, vitamins and bile acid metabolites. Some of these can also be absorbed into the systemic circulation to provide associated health benefits. The absorption of these metabolites into the systemic circulation is, in particular, believed to be linked to a healthy immune system since certain metabolites are believed to modulate immune cell function. The gut microbiota is also believed to play an important role in the gut-brain axis which is the biochemical signalling that takes place between the gastrointestinal tract and the central nervous system. The gut microbiome produces various compounds that play a role in this signalling system.

[0008] Vitamins are a type of metabolite known to be produced in the gut microbiome. In particular, it is known that the gut microbiome can produce vitamin K and B group vitamins. The production of these vitamins and their absorption into the systemic circulation is associated with various health and wellbeing benefits. Riboflavin (vitamin B2) is a vitamin produced by the gut microbiome. Riboflavin is also found naturally in a wide variety of foods such as dairy products, organ meat, eggs and green leafy vegetables. Riboflavin plays a key role in ensuring a normal energy-yielding metabolism, healthy reproductive function, lactation and successful pregnancy outcomes. It also contributes to multiple physiological processes such as energy metabolism, healthy metabolism of iron, maintenance of skin and mucous membranes, healthy red blood cells, healthy vision, protection of DNA, proteins and lipids from oxidative damage, healthy function of the nervous system, and a reduction in tiredness and fatigue. Riboflavin is thus important for human health and wellbeing. Riboflavin is produced by the gut microbiome where it is then absorbed in the large intestine and converted into the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) which have an important role in energy metabolism. Since riboflavin is not stored in the body, a continued supply is required. Both oral intake and production by the microbiota contribute to this continued supply. Colonic riboflavin is indicated to inhibit pathogen growth, increases the abundance of specific anaerobic bacteria which are connected with health (e.g. Roseburia and F.Prausnitzii). Moreover, butyrate production is directly affected by the availability of riboflavin in the colon. In conditions of microbial dysbiosis (e.g. by health status, diet, infections, antibiotics, drugs, smoking, alcohol, hygiene) the production of specific microbial metabolites, including riboflavin, can be diminished.

[0009] Pyridoxamine (vitamin B6) is another vitamin produced by the gut microbiome and then absorbed into the systemic circulation. Pyridoxamine is involved in a wide variety of enzymatic reactions related to amino acid, protein, nitrogen, glucose and lipid metabolism and hormonal activity. Pyridoxamine is found naturally in a wide variety of foods such as fish, poultry, organ meats, potatoes and other starchy vegetables and non-citrus fruits. Pyridoxamine is also associated with controlling blood pressure and clotting and immune modulation (e.g. reducing inflammation). Since pyridoxamine is, only stored in the body to a limited extent, a continued supply is required. Both oral intake and production by the microbiota contribute to this continued supply. Colonic pyridoxamine serves as an essential nutrient for specific microbiota. Moreover, higher levels are associated with greater microbial richness and evenness. In conditions of microbial dysbiosis (e.g. by health status, diet, infections, antibiotics, drugs, smoking, alcohol, hygiene) the production of specific microbial metabolites, including pyridoxamine, can be diminished.

[0010] In order to promote the positive health and wellbeing effects associated with healthy levels of vitamins such as riboflavin and pyridoxamine, supplements containing these vitamins are often taken. A problem associated with oral supplementation of these vitamins is that the effects of the supplements are not long lasting. Moreover, certain probiotic oral supplements are also known that contain vitamin producing bacteria. A problem with probiotic supplementation is that it can be unreliable since many probiotic bacteria can be destroyed by stomach acid before reaching the colon.

[0011] There thus remains a need in the art forways of increasing the amount of gut microbiome- produced vitamins so that the vitamins are available for absorption into the systemic circulation and locally to modulate microbial metabolism and composition, to promote gut health and the health and wellbeing effects discussed above.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention is based on the surprising finding that lipids can be used to stimulate the mammalian gut microbiome to produce certain vitamins. These vitamins are then available locally for modulating microbial metabolism and composition and for absorption into the systemic circulation to promote various health and wellbeing benefits such as those discussed above.

[0014] According to a first aspect of the invention, there is provided the non-therapeutic use of one or more lipids to stimulate the mammalian gut microbiome of an individual to produce one or more vitamins comprising vitamin K or one or more B group vitamins.

[0015] Preferably, the use comprises in vivo stimulation of the mammalian gut microbiome; and the use comprises administering a lipid composition comprising the one or more lipids to the individual.

[0016] In other instances, the use comprises ex vivo stimulation of the gut microbiome to produce one or more vitamins. For example, microorganisms derived from the gut microbiome of an individual may be removed from the colon of the individual (for example by taking a stool sample that may comprise microorganisms from the colon) and contacting the stool sample comprising the microorganisms, or specific isolated bacterial strains with one or more lipids. The contacting may be done under conditions suitable to cause fermentation of the one or more lipids by the microorganisms of the microbiome.

[0017] The individual is preferably a human and the gut microbiome is preferably the human gut microbiome. Alternatively, the individual may be another type of mammal comprising a gut microbiome. Examples of other types of mammal that may have their gut microbiome stimulated by the one or more lipids include livestock such as cattle, sheep and pigs; or domesticated animals and pets such as dogs and cats.

[0018] Preferably, the gut microbiome is stimulated to produce a greater amount of the one or more vitamins by the one or more lipids or lipid composition relative to the degree of stimulation by the same weight of glucose. Surprisingly, it has been found that the gut microbiome is stimulated to produce vitamins to a greater extent than the same weight of glucose. Typically, microorganisms such as bacteria ferment sugars and carbohydrate such as polysaccharides and convert these molecules into metabolites such as certain vitamins in the case of the gut microbiome. A surprising finding of the present invention is that lipids stimulate the mammalian gut microbiome to produce vitamins to a greater extent than a comparative amount of glucose.

[0019] The one or more lipids or lipid composition may comprise any type of suitable lipid. Preferably, the one or more suitable lipids or lipid composition comprises one or more free fatty acids or salts thereof; one or more fatty acid esters, or a combination thereof. Where the composition comprises one or more fatty acid esters, preferably, the one or more fatty acid esters comprise one or more triglycerides, one or more diglycerides, one or more monoglycerides; or a combination thereof. More preferably, where the composition comprises one or more fatty acid esters, the one or more fatty acid esters comprise one or more triglycerides. In other instances, the one or more fatty acid esters comprise one or more alkyl fatty acid esters, such as one or more C1 to C5 alkyl esters. Preferably, where the one or more fatty acid esters comprise one or more alkyl fatty acid esters, the one or more alkyl fatty acid esters comprise one or more ethyl fatty acid esters.

[0020] The term lipid as used herein is also used to referto molecules that comprise a lipid moiety covalently conjugated to an additional non-lipid moiety, as discussed in further detail below. The lipid moiety can be any suitable lipid moiety such as a fatty acid moiety or fatty acid glyceride moiety. The additional non-lipid moiety may be any suitable moiety. Preferably, the non-lipid moiety comprises a polysaccharide and more preferably dietary fiber. The non-lipid moiety and lipid moiety may be conjugated covalently in any suitable way such as via an ester bond. Preferably, the lipid moiety is a fatty acid moiety and the non-lipid moiety is a polysaccharide such as dietary fiber. Preferably, these moieties are conjugated via an ester bond. In some instances, the one or more lipids may be derived from or obtained from one or more plant derived triglyceride oils or one or more animal- derived triglyceride oils. Examples of plant derived triglyceride oils that may be used include high oleic sunflower oil, sunflower oil, rapeseed oil, high oleic rapeseed oil, soybean oil, linseed oil, olive oil, corn oil, cottonseed oil, groundnut oil, safflower oil, high oleic safflower oil, peanut oil, rice oil, camelina oil, sesame oil, rice bran oil, evening primrose oil, borage oil, walnut oil, palm fat, coconut fat, shea butter, cocoa butter, allanblackia fat, kokum fat, mango kernel fat, sal fat, illipe butter, palm kernel fat, babassu fat, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, any fractions thereof , any interesterified forms thereof, or any combinations thereof. Examples of one or more animal derived triglyceride oils that may be used include tallow, lard, poultry fat, fish oil, milk fat, any fractions thereof, any interesterified forms thereof, or a combination thereof. The one or more lipids or lipid composition may alternatively or additionally be derived from or obtained from one or more microbially derived oils such as algae oil. In some instances, the lipid composition may comprise these oils themselves.

[0021] Preferably, the one or more lipids or lipid composition comprises one or more free fatty acids or salts thereof. The one or more free fatty acids or salts thereof may be derived from or obtained from any suitable lipid source such as those discussed above. In some instances, the one or more free fatty acids may be derived from fermentation by one or more microorganisms. Preferably, the one or more microorganisms comprise one or more bacterial species, one or more yeast species, ora combination thereof. In some instances, the one or more microorganisms such as the one or more bacterial species or one or more yeast species are genetically modified.

[0022] Typically, the one or more lipids or lipid composition comprises at least 50% by weight of free fatty acids or salts thereof. Preferably, the one or more lipids or lipid composition comprises at least 60% by weight of free fatty acids or salts thereof. More preferably, the one or more lipids or lipid composition comprises at least 70% by weight of free fatty acids or salts thereof . Still more preferably, the one or more lipids or lipid composition comprises at least 80% by weight of free fatty acids or salts thereof. Most preferably, the one or more lipids or lipid composition comprises at least 90% by weight of free fatty acids or salts thereof.

[0023] The one or more free fatty acids may comprise any suitable free fatty acid or salts thereof. For example, the one or more free fatty acids or salts thereof may comprise freefatty acids or salts thereof derived from or obtained from any one or more of the animal or plant derived triglyceride oils described above.

[0024] Typically, the one or more lipids or lipid composition comprise free fatty acids having 18 carbon atoms or 12 carbon atoms, salts thereof, esters thereof, or any combination thereof. Where the one or more lipids or lipid composition comprises one or more esters of free fatty acids, preferably, the esters comprise glyceride esters and more preferably triglyceride esters.

[0025] Preferably, the one or more lipids or lipid composition comprise lauric acid (C12:0), oleic acid (C18: 1 ), linoleic acid (C18:2), stearic acid (C18:0), salts thereof, esters thereof, or a combination thereof. Where the one or more lipids or lipid composition comprises one or more esters of these free fatty acids, preferably, the esters comprise glyceride esters and more preferably triglyceride esters.

[0026] More preferably, the one or more lipids or lipid composition comprise lauric acid (C12:0), oleic acid (C18:1 ), linoleic acid (C18:2), salts thereof, esters thereof, or a combination thereof. Where the one or more lipids or lipid composition comprises one or more esters of these free fatty acids, preferably, the esters comprise glyceride esters and more preferably triglyceride esters.

[0027] Typically, non-therapeutic use according to the invention comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises at least 40% by weight, and preferably at least 50% by weight, of a total of lauric acid (C12:0), oleic acid (C18:1 ) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, freefatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

[0028] More preferably, the use comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises at least 60% by weight, and still more preferably at least 70% by weight, of a total of lauric acid (C12:0), oleic acid (C18:1 ) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

[0029] Most preferably, the use comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises at least 80% by weight, of a total of lauric acid (C12:0), oleic acid (C18: 1 ) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

[0030] Non-therapeutic use according to any preceding claim, wherein the one or more lipids or lipid composition comprises pentadecanoic acid (C15:0) and / or heptadecanoic acid (C17:0), salts thereof, esters thereof such as triglyceride, diglyceride or monoglyceride esters thereof. A source for pentadecanoic acid (C15:0) is dairy fat, which typically contains 1 to 3% C15:0. Heptadecanoic acid (C17:0) occurs in small amounts (typically <1 %) in dairy fat, as well as the fat of ruminants.

[0031] More preferably, the composition comprising C15:0 also comprises at least one of lauric acid (C12:0), oleic acid (C18:1) and linoleic acid (C18:2). Even more preferably, the composition comprises a combination of lauric acid (C12:0), pentadecanoic acid (C15:0), heptadecanoic acid (C17:0), oleic acid (C18:1 ) and linoleic acid (C18:2).

[0032] The one or more vitamins produced by the gut microbiome may comprise vitamin K or one or more B group vitamins. These vitamins are produced naturally by the gut microbiome as a result of their metabolism.

[0033] The term vitamin as used herein is given its normal meaning in the art to refer to a group of organic molecules that are essential to a mammalian organism in small quantities for proper metabolic function. Examples of vitamins include Vitamin A, Vitamin Bi (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (Niacin), Vitamin Bs (pantothenic acid), Vitamin Be (pyridoxamine), Vitamin B? (biotin), Vitamin Bg (folic acid and folates), Vitamin B12 (cobalamines), Vitamin C (ascorbic acid), Vitamin D (calciferols), Vitamin E (tocopherols and tocotrienols) and Vitamin K. The term vitamin is used to refer to neutral vitamin molecules and also any salts or ionic forms thereof, including any zwitterionic forms of the vitamin.

[0034] The one or more vitamins produced by the gut microbiome comprise vitamin K or one or more B group vitamins. Preferably, the one or more vitamins comprise one or more B group vitamins. More preferably, the one or more B group vitamins comprise Vitamin Bi (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (Niacin), Vitamin Bs (pantothenic acid), Vitamin Be (pyridoxamine), Vitamin B? (biotin), Vitamin Bg (folicacid and folates), Vitamin B12 (cobalamines), or any combination thereof. More preferably, the one or more vitamins produced by the gut microbiome comprise riboflavin, pyridoxamine, or a combination thereof.

[0035] Where the one or more vitamins comprise riboflavin, it has surprisingly been found that the gut microbiome is stimulated to produce this vitamin by fatty acids having 12 carbon atoms to a greater extent than other lipids and in particular other fatty acids containing more or less carbon atoms. In particular, it has surprisingly been found that lauric acid (C12:0) stimulates the gut microbiome to produce riboflavin to a greater extent than other lipids such as different fatty acids.

[0036] Accordingly, preferably, where the one or more vitamins comprise riboflavin, the one or more lipids or lipid composition comprises one or more free fatty acids having 12 carbon atoms, salts thereof or esters thereof. Where the one or more lipids or lipid composition comprises one or more esters of these free fatty acids, preferably, the esters comprise glyceride esters and more preferably triglyceride esters.

[0037] More preferably, where the one or more vitamins comprise riboflavin, the one or more lipids or lipid composition comprises lauric acid (C12:0), salts thereof, esters thereof, or a combination thereof. Where the one or more lipids or lipid composition comprises one or more esters of these free fatty acids, preferably, the esters comprise glyceride esters and more preferably triglyceride esters. However, as discussed above, preferably, the one or more lipids or lipid composition comprises free fatty acids.

[0038] Typically, where the one or more vitamins comprise riboflavin, the use comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises at least 40% by weight, and preferably at least 50% by weight, of lauric acid (C12:0), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

[0039] More preferably, where the one or more vitamins comprise riboflavin, the use comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises at least 60% by weight, and still more preferably at least 70% by weight, of lauric acid (C12:0), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

[0040] Still more preferably, where the one or more vitamins comprise riboflavin, the use comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises at least 80% by weight, and most preferably at least 90% by weight of lauric acid (C12:0), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition , free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

[0041] Where the one or more vitamins comprise riboflavin, the one or more lipids or lipid composition can comprise any suitable lipid such as those discussed above. It is preferable that the one or more lipids or lipid composition comprises one or more lipids that are high in fatty acids having 12 carbon atoms, and more preferably that the one or more lipids or lipid composition comprises one or more lipids that are high in lauric acid (C12:0). For example, the one or more lipids or lipid composition may be derived from or obtained from medium chain triglyceride (MCT) oil that is high in lauric acid. MCT oil can be extracted from a variety of sources such as coconut oil, palm kernel oil, and certain types of milk. Methods for extraction of MCT oil will be apparent to the skilled person given the benefit of the present disclosure. The one or more lipids or lipid composition may also be derived from or obtained from one or more fats or oil that is high in lauric acid such as lauric fats or milk fat. Examples of lauric fats include coconut oil, palm kernel oil, babassu oil, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, any fraction thereof, any interesterified form thereof, or any combination thereof. In some instances, the lipid composition may comprise the abovementioned oils themselves. In these instances, preferably, the abovementioned oils are interesterified, such as chemically interesterified or enzymatically interesterified. Interesterification such as chemical interesterification provides a more random distribution of triglycerides while enzymatic interesterification can provide a more particular distribution of triglycerides within the oil. Interesterification may be used to alter the positions of the lauric acid (C12:0) residues in the triglycerides to promote delivery of the lauric acid (C12:0) to the colon as discussed in further detail below.

[0042] As discussed above, it is preferable that the one or more lipids or lipid composition comprises free fatty acids or salts thereof. Accordingly, preferably, the use comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises free fatty acids derived from or obtained from medium chain triglyceride (MCT) oil, coconut oil, palm kernel oil, babassu oil, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, milk fat, any fraction thereof, or any combination thereof. Methods of obtaining free fatty acids from such oils will be apparent to the skilled person given the benefit of the present disclosure. As discussed above, the free fatty acids may also be derived from fermentation by one or more microorganisms.

[0043] Where the one or more vitamins comprise pyridoxamine, it has surprisingly been found that the gut microbiome is stimulated to produce this vitamin by fatty acids having 18 carbon atoms in length to a greater extent than other lipids and in particular other fatty acids containing more or less carbon atoms. In particular, it has been found that oleic acid (C18:1) and linoleic acid (C18:2) stimulate the gut microbiome to produce pyridoxamine to a greater extent than other lipids such as different fatty acids.

[0044] Accordingly, where the one or more vitamins comprise pyridoxamine, preferably, the one or more lipids or lipid composition comprises one or more free fatty acids having 18 carbon atoms, salts thereof or esters thereof. Where the one or more lipids or lipid composition comprises one or more esters of these free fatty acids, preferably, the esters comprise glyceride esters and more preferably triglyceride esters. However, as discussed above, preferably, the one or more lipids or lipid composition comprises free fatty acids.

[0045] More preferably, where the one or more vitamins comprise pyridoxamine, the one or more lipids or lipid composition comprises oleic acid (C18:1 ), linoleic acid (C18:2), salts thereof, esters thereof or a combination thereof. Where the one or more lipids or lipid composition comprises one or more esters of these free fatty acids, preferably, the esters comprise glyceride esters and more preferably triglyceride esters. However, as discussed above, preferably, the one or more lipids or lipid composition comprises free fatty acids.

[0046] Typically, where the one or more vitamins comprise pyridoxamine, the use comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises at least 70% by weight of a total of oleic acid (C18:1) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

[0047] Preferably, where the one or more vitamins comprise pyridoxamine, the use comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises at least 80% by weight of a total of oleic acid (C18:1) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

[0048] More preferably, where the one or more vitamins comprise pyridoxamine, the use comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises at least 90% by weight of a total of oleic acid (C18:1 ) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

[0049] Where the one or more vitamins comprise pyridoxamine, the one or more lipids or lipid composition can comprise any suitable lipid such as those discussed above. It is preferable that the one or more lipids or lipid composition is derived from or obtained from a source of lipids that is high in fatty acids having 18 carbon atoms, and more preferably that the one or more lipids or lipid composition is derived from or obtained from a source of lipids that are high in of oleic acid (C18: 1 ) and linoleic acid (C18:2). Examples of such lipid sources include soyabean oil, sunflower oil, rapeseed oil, groundnut oil, cottonseed oil, maize oil, safflower oil, sesame oil, rice bran oil, evening primrose oil, borage oil, palm oil, any fraction thereof, or any combination thereof. As discussed above, where the one or more lipids comprise free fatty acids, the free fatty acids may also be derived from fermentation by one or more microorganisms.

[0050] In some instances, the lipid composition may comprise these oils themselves. In these instances, preferably, the abovementioned oils are interesterified, such as chemically interesterified or enzymatically interesterified. Interesterification such as chemical interesterification provides a more random distribution of triglycerides within the oil which may be used to alter the positions of the fatty acid residues in the triglycerides to promote delivery of the C18 fatty acids such as oleic acid to the colon as discussed in further detail below.

[0051] As discussed above, it is preferable that the one or more lipids or lipid composition comprises free fatty acids or salts thereof. Accordingly, preferably, the use comprises administering a lipid composition comprising the one or more lipids to an individual; where the lipid composition comprises free fatty acids derived from soyabean oil, sunflower oil, rapeseed oil, groundnut oil, cottonseed oil, maize oil, safflower oil, sesame oil, rice bran oil, evening primrose oil, borage oil, palm oil, any fraction thereof, or any combination thereof. Methods of obtaining free fatty acids from such oils will be apparent to the skilled person given the benefit of the present disclosure. As discussed above, the free fatty acids may also be derived from fermentation by one or more microorganisms.

[0052] In some instances, it may be desirable to administer to the individual a lipid composition that comprises a variety of different lipids that have each been found to stimulate the mammalian gut microbiome to produce different desired vitamins. For example, it may be desirable to stimulate the gut microbiome to produce both riboflavin, pyridoxamine, and / or other vitamins such as other B group vitamins. In these instances, for example, it may be desirable to administer a lipid composition that comprises two or more of (i) oleic acid (C18:1) or salts or esters thereof, (ii) linoleic acid (C18:2) or salts or esters thereof, and (iii) lauric acid (C12:0) or salts or esters thereof. Preferably, the use may comprise administering a lipid composition high in all three of these fatty acids or salts or esters thereof.

[0053] For example, the lipid composition may comprise soyabean oil, sunflower oil, rapeseed oil, groundnut oil, cottonseed oil, maize oil, safflower oil, sesame oil, rice bran oil, evening primrose oil, borage oil, palm oil, coconut oil, palm kernel oil, babassu oil, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, milk fat, any fraction thereof, any interesterified form thereof, or any combination thereof.

[0054] For example, the lipid composition may be derived or obtained from (i) soyabean oil, sunflower oil, rapeseed oil, groundnut oil, cottonseed oil, maize oil, safflower oil, sesame oil, rice bran oil, evening primrose oil, borage oil, palm oil, any fraction thereof, or any combination thereof; and (ii) coconut oil, palm kernel oil, babassu oil, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, milk fat, any fraction thereof, any interesterified form thereof, or any combination thereof. In some instances, the lipid composition may comprise these oils themselves. As discussed above, in these instances, it is preferred that the oils are interesterified such as chemically interesterified or enzymatically interesterified.

[0055] Since the lipid composition is preferably high in free fatty acids, preferably, the lipid composition comprises free fatty acids derived from soyabean oil, sunflower oil, rapeseed oil, groundnut oil, cottonseed oil, maize oil, safflower oil, sesame oil, rice bran oil, evening primrose oil, borage oil, palm oil, coconut oil, palm kernel oil, babassu oil, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, milk fat, any fraction thereof, or any combination thereof. As discussed above, the free fatty acids may also be derived from fermentation by one or more microorganisms.

[0056] For example, the lipid composition may comprise (i) free fatty acids derived from soyabean oil, sunflower oil, rapeseed oil, groundnut oil, cottonseed oil, maize oil, safflower oil, sesame oil, rice bran oil, evening primrose oil, borage oil, palm oil, any fraction thereof, or any combination thereof; and (ii) free fatty acids derived from coconut oil, palm kernel oil, babassu oil, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, milk fat, any fraction thereof, or any combination thereof.

[0057] In the instances described above, the oils and free fatty acids of component (i) are typically high in oleic acid (C18:1 ) and linoleic acid (C18:2) and the oils and free fatty acids of component (ii) are typically high in lauric acid (C12:0). Accordingly, the lipid compositions described comprise lipids that have been found to be particularly good at stimulating the gut microbiome to produce one or more B group vitamins such as both riboflavin and pyridoxamine. Such lipid compositions may thus be particularly useful for promoting the health benefits discussed above associated with production of these vitamins by the gut microbiome. The use can comprise administration of any suitable amount of lipid for stimulating the gut microbiome to produce the one or more vitamins.

[0058] Preferably, the use comprises administration of a lipid composition comprising one or more lipids to the individual; and the lipid composition comprises from 0.5 grams to 10 grams of the one or more lipids per serving; and preferably from 1 to 5 grams of the one or more lipids per serving.

[0059] More preferably, the lipid composition comprises from 0.5 grams to 10 grams of a total of lauric acid (C12:0), oleic acid (C18:1 ), linoleic acid (C18:2), and salts and esters thereof per serving; and preferably from 1 gram to 5 grams of a total of lauric acid (C12:0), oleic acid (C18:1), linoleic acid (C18:2), and salts and esters thereof per serving.

[0060] In some instances, the lipid composition is a unit dosage form. Any suitable unit dosage form may be used such as capsules, pills, or tablets etc. where the lipid composition is administered orally. Typically, where the lipid composition is in the form of a unit dosage form, the lipid composition is a unit dosage form comprising from 0.5 grams to 10 grams of the one or more lipids. Preferably, where the lipid composition is a unit dosage form, the unit dosage form comprises from 1 gram to 5 grams of a total of lauric acid (C12:0), oleic acid (C18:1 ), linoleic acid (C18:2), and salts and esters thereof.

[0061] Where the one or more vitamins comprise riboflavin, the lipid composition preferably comprises from 0.5 grams to 10 grams, preferably from 1 gram to 5 grams of a total of lauric acid (C12:0) and salts and esters thereof per serving.

[0062] Where the one or more vitamins comprise pyridoxamine; the lipid composition preferably comprises from 0.5 grams to 10 grams, preferably from 1 gram to 5 grams of a total of oleic acid (C18:1 ) and linoleic acid (C18:2), and salts and esters thereof per serving.

[0063] The one or more lipids are typically administered to an individual in a daily dose that is sufficient to stimulate the gut microbiome to produce the one or more vitamins.

[0064] Typically, the use comprises administration of from 0.5 grams to 10 grams, preferably from 1 gram to 5 grams per day of the one or more lipids to the individual. Preferably, the use comprises administration of from 0.5 grams to 10 grams, preferably from 1 gram to 5 grams per day of a total of lauric acid (C12:0), oleic acid (C18:1), linoleic acid (C18:2), and salts and esters thereof to the individual.

[0065] In some instances, the use comprises administration of from 0.5 grams to 10 grams, preferably from 1 gram to 5 grams per day of a total of lauric acid (C12:0), and salts and esters thereof to the individual. Such a dosage may be particularly preferred where it is desired for the microbiome to be stimulated to produce riboflavin.

[0066] In other instances, the use comprises administration of from 0.5 grams to 10 grams, preferably from 1 gram to 5 grams per day of a total of oleic acid (C18:1 ), linoleic acid (C18:2), and salts and esters thereof to the individual. Such a dosage may be particularly preferred where it is desired forthe microbiome to be stimulated to produce pyridoxamine.

[0067] The administration of the one or more lipids may be carried out for any duration of time needed for the microbiome to be stimulated to produce the one or more vitamins. Typically, the one or more lipids or lipid composition may be administered to an individual for a time period of at least one day; preferably at least one week; more preferably at least one month; still more preferably at least six months; and most preferably at least one year. For example, the administration of the one or more lipids may be carried out for a time period of from one day to five years, one week to five years, one month to five years, one month to one year, one month to six months or one month to three months.

[0068] The lipid composition may be formulated in any suitable manner for delivery of the lipid composition to the gut microbiome.

[0069] Since the gut microbiome is predominantly present in the distal part of the small intestine and the colon of mammals, preferably, the use comprises administering the lipid composition in a manner such that the one or more lipids of the lipid composition reach the distal part of the small intestine and colon of the mammal without prior absorption from the intestinal tract.

[0070] Accordingly, preferably, the lipid composition is formulated for delivery to the distal part of the intestines, and preferably colonic delivery of the one or more lipids.

[0071] Preferably, the lipid composition is formulated for oral administration where the lipid composition is administered to the individual by oral administration. In alternative embodiments, the lipid composition may be administered in a different manner such that the lipid composition is delivered to the colon. For example, the lipid composition may be formulated for rectal administration and administered to the individual by rectal administration.

[0072] Preferably, the lipid composition is formulated for colonic delivery of the one or more lipids; the lipid composition is formulated for oral administration and the use comprises oral administration of the lipid composition to the individual. Any suitable means of formulation for colonic delivery and oral administration known in the art may be used forthis purpose. For example, a wide variety of formulation techniques for colonic delivery of an active pharmaceutical or nutraceutical ingredient are discussed in the article, Colon Targeted Drug Delivery Systems: A Review on Primary and Novel Approaches. Philip et Al., Oman Med J. 2010 Apr; 25 (2): 79-87. Any suitable method discussed in this document may be used, the disclosure of which is incorporated herein.

[0073] In some instances, the lipid composition is encapsulated with one or more of a pH sensitive polymer; a delayed release polymer; a polymer that is degradable by one or more microbes of the mammalian gut microbiome, or a combination thereof. Formulating the lipid composition in this manner may be useful for delivery of the one or more lipids of the lipid composition to the colon. In the case of pH sensitive polymers, the pH of the colon is different to that of the pH higher up in the gastrointestinal tract such as in the small intestine or stomach. Polymers can be chosen to encapsulate the lipid compositions that do not degrade in the stomach or small intestine but that degrade at colonic pH to release the one or more lipids so that they contact the microorganisms of the gut microbiome present in the colon. Typically, the pH sensitive polymer is selected to start dissolving at pH of around 6 to 7 which is the pH of the terminal ileum and colon and be insoluble at the lower pH values found in the stomach and proximal region of the small intestine. In the case of delayed release polymers, a polymer can be chosen that takes a period of time to degrade that it takes a typical orally administered composition to pass from the stomach, through the small intestine and eventually to the colon. Polymers may also be chosen that are caused to degrade by contact with the one or more microbes of the gut microbiome, substances secreted by such microbes, or enzymes found within the colon. In such cases, the polymers may not degrade until contact with the gut microbiome or enzymes in the colon whereupon the polymer degrades such that the lipid composition contacts the gut microbiome. In other instances, polymers that degrade under pressure may be used. For example, it is known to use polymers that degrade as a result of the pressure caused by the peristaltic movement of the gastrointestinal tract to degrade polymer coatings to release ingredients encapsulated therein at a desired location.

[0074] Examples of polymers that can be used to encapsulate the lipid composition include any suitable polymer known in the art. Examples of such polymers include one or more of the following polymers: cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate trimellitate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, and copolymers of two or more of acrylic acid, methacrylic acid, salts thereof and esters thereof. Examples of copolymers of two or more of acrylic acid, methacrylic acid, salts thereof and esters thereof include copolymers of two or more of methyl acrylate, methyl methacrylate, methacrylic acid and salts thereof and ethyl methacrylate. Examples of such polymers are commercially available under the trade name Eudragit® and will be apparent to the skilled person given the benefit of the present disclosure. Specific examples include Eudragit® FS, Eudragit® S, Eudragit® L, and Eudragit® FL, and Eudragit® FL 30 D-55.

[0075] The polymer coatings should also be sufficiently thick to ensure that the encapsulated lipid cargo is not released until distal part of the small intestine, more preferably the colon. Suitable coating thicknesses for a given polymer will be apparent to the skilled person given the benefit of the present disclosure.

[0076] In other instances, the lipid composition may be coated with one or more polysaccharides. Examples of such polysaccharides include chitosan, pectin, starch, chondroitin and salts thereof , alginate, gums, amylose, dextran, inulin, galactomannan, cyclodextrins, cellulose, derivatives thereof, or combinations thereof. Commercially available examples of such polysaccharides will be known to the skilled person given the benefit of the present disclosure and include Eudragit® S100 and Eudragit® L100 t.

[0077] In other instances, the one or more lipids of the lipid composition may be dispersed within a delayed release matrix that does not release the one or more lipids until they are delivered to the distal part of the small intestine, and more preferably the colon. Delayed release matrixes can take the form of delayed release tablets that can be swallowed where the lipid composition is dispersed within a delayed release matrix such as a delayed release polymer. Alternatively, the one or more lipids may be dispersed in an edible delayed release matrix that be chewed and swallowed by the individual but that does not break down fully to release the one or more lipids until delivery to the colon. Examples of such edible delayed release matrixes and matrixes that can be used in delayed release tablets include matrixes comprising or formed from the polysaccharide polymers discussed above in the context of polysaccharide coatings.

[0078] Polymers used in edible delayed release matrixes, delayed release tablets or coatings such as the polysaccharide coatings discussed above may be degraded by colonic pH or when contacted with substances secreted by the colon that degrade the polymers, the colonic microbiome, or enzymes found within the colon (for example enzymes secreted by the colon or enzymes present in the cell membranes of colon tissue). In other instances, the one or more lipids of the lipid composition may be encapsulated within an oleosome. The term oleosome as used herein is used to refer to a vesicle with a diameter of typically from 0.1 pm to 10 pm that comprises a monolayer of phospholipids that is embedded with a layer of oleosin (a type of plant protein), encapsulating an interior. The oleosomes can be stabilized by an extra capsule / coating of hydrocolloids / dextrin if desired. The interior of the oleosome vesicle may comprise cargo such as the one or more lipids or lipid composition used in the present invention. The one or more lipids or lipid composition are thus encapsulated by the phospholipid monolayer of the oleosome. Methods of preparing oleosomes comprising the one or more lipids or lipid composition will be apparent to the skilled person given the benefit of the present disclosure. The oleosomes are only partially broken down by the conditions of the stomach and proximal small intestine meaning that the content of the oleosomes is delivered to the distal part of the small intestine and the colon. The oleosomes are digested at a relatively slow rate enabling the release the one or more lipids or lipid composition such that it contacts the colonic gut microbiome.

[0079] In other instances, delivery of the one or more lipids to the colon may comprise conjugating the one or more lipids to a chemical moiety that prevents breakdown of the one or more lipids in the gastrointestinal tract until the lipid composition reaches the colon. For example, the lipid composition or one or more lipids may be conjugated to a fiber component. Fiber comprises plant-derived polysaccharide molecules that are not digested by the stomach or small intestine and so pass directly to the colon. Once in the colon, enzymes degrade the covalent bond between the one or more lipids and the chemical moiety such as fiber and the one or more lipids thus contact the gut microbiome within the colon. Examples of suitable fibers, other chemical moieties and conjugation chemistries will be apparent to the skilled person given the benefit of the present disclosure. As discussed above, the term lipid thus encompasses molecules that comprise a lipid moiety covalently conjugated to an additional moiety.

[0080] In other instances, specific lipid molecules themselves may be selected that are not degraded or absorbed higher up in the gastrointestinal tract and that as such find their way to the distal part of the small intestine and the colon so as to contact the colonic gut microbiome. For example, it is known that lipase enzymes higher up in the gastrointestinal tract such as pancreatic lipase preferentially hydrolyse fatty acids bound at the Sn-3 and Sn-1 position over fatty acids bound at the Sn-2 position of glycerol in triglyceride molecules. However, it is also known that the various lipases preferentially hydrolyse saturated fatty acids over monounsaturated and polyunsaturated fatty acids bound in triglycerides. The selectivity of the lipases thus promote the release of saturated free fatty acids and MAG . The upper gastrointestinal tract is adapted to preferentially absorb these hydrolysis products and preferentially absorbs MAG with a fatty acid bound at the Sn-2 position over the free fatty acids. Undigested triacylglycerol (TAG) will not be absorbed while the partially digested diacylglycerol (DAG) are taken up by the body only limited. They are not preferentially absorbed by the upper gastrointestinal tract, meaning that they pass to the distal part of the small intestine and the colon Considering this, delivery of certain unsaturated fatty acids (such as the preferred C18:1 and C18:2) to the colon can be promoted by using (structured) triglycerides that have unsaturated fatty acids (such as the preferred C18:1 and C18:2) bound at the Sn-1 or Sn-3 positions. These MAG and DAG.

[0081] Triglycerides with unsaturated fatty acids (such as C18:1 or C18:2) bound at the Sn-1 or Sn-3 positions may occur naturally in certain oils such as vegetable oils. For example, the triglycerides POO and PLO (with oleic acid at the Sn-3 position) are found in palm oil. Alternatively, triglycerides with unsaturated fatty acids at the Sn-1 or Sn-3 positions may be formed by interesterification of vegetable oils to promote the formation of the desired triglycerides.

[0082] In some instances, delivery of the lipids to the distal part of the small intestine and the colon can be achieved by combining different delivery systems.

[0083] According to a second aspect of the invention, there is provided a nutraceutical product comprising a lipid composition comprising one or more lipids, wherein the lipid composition comprises lauric acid (C12:0), oleic acid (C18: 1 ), linoleic acid (C18:2), salts thereof , esters thereof, or a combination thereof.

[0084] Preferably, the lipid composition is formulated forcolonic delivery of the one or more lipids.

[0085] Preferably, the lipid composition is as described above in accordance with the first aspect of the invention.

[0086] Preferably, the nutraceutical product comprises a lipid composition comprising one or more lipids, wherein the lipid composition comprises lauric acid (012:0), oleic acid (C18:1 ) and linoleic acid (C18:2). The lipid composition can be present in the nutraceutical product in any suitable amount. Typically, the nutraceutical product comprises the lipid composition in an amount of from 0.5% to 100% by weight of the nutraceutical product; and preferably from 0.5% to 80% by weight of the nutraceutical product.

[0087] Typically, the nutraceutical product comprises from 0.5 grams to 10 grams of a total of lauric acid (C12:0), oleic acid (C18:1 ), linoleic acid (C18:2), and salts and esters thereof per serving, and preferably, from 1 gram to 5 grams of a total of lauric acid (C12:0), capric acid (C10:0), oleic acid (C18:1), linoleic acid (C18:2), and salts and esters thereof per serving.

[0088] In instances where it is desired for the gut microbiome to be stimulated to produce riboflavin, the nutraceutical product typically comprises from 0.5 grams to 10 grams of a total of lauric acid (C12:0), and salts and esters thereof per serving, and preferably, from 1 gram to 5 grams of a total of oleic acid (C18:1 ) and linoleic acid (C18:2), and salts and esters thereof per serving.

[0089] Odd-chain fatty acids found to stimulate riboflavin production are pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0).

[0090] In instances where it is desired for the gut microbiome to be stimulated to produce pyridoxamine, the nutraceutical product typically comprises from 0.5 grams to 10 grams of a total of oleic acid (C18:1) and linoleic acid (C18:2), and salts and esters thereof per serving, and preferably, from 1 gram to 5 grams of a total of lauric acid (C12:0), capric acid (C10:0), and salts and esters thereof per serving.

[0091] An odd-chain fatty acid that was found to stimulate pyridoxamine production is heptadecanoic acid (C17:0).

[0092] The nutraceutical product can be in any suitable form. For example, the nutraceutical product can be in a solid form such as a tablet or capsule and swallowed by the individual. In other instances, a powder of the nutraceutical product may be provided that is designed to be mixed with a liquid such as milk or water to form a solution or suspension that is then drunk by the individual. In other instances, the nutraceutical product may be in a liquid form such as a drink. In other instances, the nutraceutical product may be a functional food, for example a functional food comprising a delayed release matrix of the lipid composition designed to deliver the lipid composition to the colon. Where the nutraceutical product is in solid form, the product is typically a powder, tablet, dough, bar or capsule. In these instances, the product typically comprises the lipid composition in an amount of from 5% to 100% by weight; preferably from 5% to 95% by weight; more preferably from 20% to 85% by weight; still more preferably from 35% to 75% by weight; and most preferably from 50% to 80% by weight. In other instances, the product may comprise the lipid composition in an amount of from 50% to 95% by weight such as 50% to 90% by weight or 50% to 80% by weight. In other instances, the product may comprise the lipid composition in an amount of from 5% to 50% by weight such as from 10% to 50% by weight or 20% to 40% by weight.

[0093] Where the nutraceutical product is in solid form, suitable carriers, excipients and other functional food ingredients to be included in the compositions will be apparent to the skilled person given the benefit of the present disclosure.

[0094] In some instances, the nutraceutical product is a food product or an infant formula. In such cases, the nutraceutical product may be either a liquid product or a solid product such as a powder that is designed to be mixed with water to form a drinkable solution or suspension.

[0095] In some instances, the nutraceutical product may be a unit dosage form such as pill or capsule or a liquid unit dosage form such as a pre-measured volume of solution or suspension of the lipid composition.

[0096] In some instances, the nutraceutical product is a unit dosage form comprising from 0.5 grams to 10 grams of a total of lauric acid (C12:0), oleic acid (C18:1 ), linoleic acid (C18:2), and salts and esters thereof, and preferably, from 1 gram to 5 grams of a total of lauric acid (C12:0), capric acid (C10:0), and salts and esters thereof per serving.

[0097] In some instances, the nutraceutical product is in a liquid form. For example, the nutraceutical product may be in the form of a drink, yoghurt or cream.

[0098] In instances where the nutraceutical product is in a liquid form, the nutraceutical product preferably comprises the lipid composition in an amount of from 0.5% to 10% by weight, such as from 1 % to 10% by weight or from 2% to 8% by weight. In other instances, the nutraceutical product comprises the lipid composition in an amount of from 0.1 % to 40% by weight such as from 1 % to 20% by weight. The nutraceutical products of the invention may be prepared using processes known in the art that will be apparent to the skilled person given the benefit of the present disclosure.

[0099] DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 depicts the concentration of riboflavin following the fermentation of fatty acids by stool samples from humans.

[0101] Figure 2 depicts the concentration of pyridoxamine following the fermentation of fatty acids by stool samples from humans.

[0102] DETAILED DESCRIPTION OF THE INVENTION

[0103] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0104] Example 1

[0105] Fecal samples from 10 donors (5 male and 5 female) were collected, mixed with one another and homogenized. A fecal slurry was prepared with 10% (wt / vol) faeces in phosphate buffered saline that contained 20% (wt / vol) glycerol and 0.05% (wt / vol) cysteine.

[0106] These samples were frozen until use before being defrosted. The prepared fecal samples were diluted 10x in a standard culture medium to study the effects of different lipids (1gi7 liter) in anaerobic culturing conditions. The culture medium contained the following components: Yeast extract (3g / l); bile salts (1 g / l); CaCl2.2H2O (0.2 g / l); FeSO4.7H2O (0.009 g / l); K2HPO4 (0.5 g / l); KCI (4.5 g / l); Tween 80 (1 ml / l); Haemin (1 g / l); Cysteine (0.5 g / l); Pancreatin (0.1 g / l); and 1 ml vitamin mix. The vitamin mix comprised the following ingredients: Folic acid (2 mg / l); Pyridoxine hydrochloride (10 mg / l); Riboflavin (5 mg / l); Biotin (2 mg / l); Thiamine (5 mg / l); Nicotinic acid (5 mg / l); calcium pantothenate (5 mg / l); vitamin B12 (0.1 mg / l); p-aminobenzoic acid (5 mg / l)l; thioctic acid (5 mg / l); and monopotassium phosphate (900 mg / l).

[0107] The following lipids were tested:

[0108] 1. Negative control (no added lipids or carbohydrates)

[0109] 2. Positive control (glucose)

[0110] 3. Butyric acid (C4:0)

[0111] 4. Caproic acid (C6:0)

[0112] 5. Caprylic acid (C8:0) 6. Capric acid (C10:0)

[0113] 7. Lauric acid (C12:0)

[0114] 8. Palmitic acid (C16:0)

[0115] 9. Stearic acid (C18:0)

[0116] 10. Oleic acid (C18: 1 )

[0117] 11 . Linoleic acid (C18:2)

[0118] 12. Alpha linolenic acid (ALA)

[0119] 13. Gamma-linolenic acid (GLA)

[0120] 14. Eicosapentaenoic acid (C20:5)

[0121] 15. Docosahexanoic acid (C22:6)

[0122] 16. Conjugated linolenic acid (CLA)

[0123] 17. Pentadecanoic acid (C15:0)

[0124] 18. Heptadecanoic acid (C17:0)

[0125] All lipids were tested in the form of fatty acids.

[0126] The fermentation of the fatty acids with the fecal samples was performed in an anaerobic system with a controlled gas atmosphere (N2 80%, H2 10% and CO2 10%), which allows mimicking of anaerobic intestinal environmental conditions. A constant temperature of 37°C was used which is human body temperature. Samples were taken every 24 hours afterthe start of incubation to measure the pH of the samples and after 72 hours of incubation at 37°C the samples were processed for further evaluation. The incubation was performed in quadruplicate.

[0127] Metabolite quantification

[0128] Cell-free supernatantsfrom the faecal batch cultures were filtered through 0.2-pm filters and stored at -80°C until analysis. The amounts of riboflavin and pyridoxamine were then quantified in the supernatants of each test condition with the following procedure.

[0129] Samples were thawed at room temperature, vortexed for 15 seconds and then centrifuged at 10000 x g for 10 minutes at 4 °C. 50 pL of each sample was placed into 1.5 mL eppendorf tubes and 350 pL of cold water was added to each sample. These mixtures were vortexed for 15 seconds. 50 pL of each sample was then transferred to a 96-well plate and mixed with 50 pL aqueous ammonium acetate (10 mM), 0.1 % v / v HCOOH:CH3CN, and 0.1 % v / v HCOOH (90:10) for UPLC-MSMS analysis. For quantification, an external calibration was carried out using standard solutions obtained from serial dilutions of a working solution containing mixtures of pure analytical standards in mixtures of aqueous ammonium acetate (10 mM), 0.1 % v / v HCOOH:CHsCN and 0.1 % v / v HCOOH (50:50).

[0130] The results for riboflavin are shown in Figure 1 and the results for pyridoxamine in Figure 2.

[0131] As can be seen in Figure 1 , relative to the glucose control, riboflavin concentration was higher for lauric acid (C12:0). Riboflavin concentration was also higher for the lauric acid sample when compared to the other free fatty acid samples tested. The results of this experiment thus show that the gut microbiome bacteria (present in the faeces) can be stimulated to produce riboflavin by lauric acid (C12:0).

[0132] Figure 1 shows that for the odd-chain fatty acids, pentadecanoic acid (C15:0) stimulates riboflavin at a level close to the positive control glucose, whereas heptadecanoic acid (C17:0) displays an even stronger stimulation of riboflavin production.

[0133] As can be seen in Figure 2, relative to the glucose control, pyridoxamine concentration was higher for oleic acid (C18:1 ) and linoleic acid (C18:2). Pyridoxamine concentration was also higher for these samples when compared to the other free fatty acid samples tested. The results of this experiment thus show that the gut microbiome bacteria (present in the faeces) can be stimulated to produce pyridoxamine by oleic acid (C18:1) and linoleic acid (C18:2).

[0134] Figure 2 shows that for the odd-chain fatty acids, heptadecanoic acid (C17:0) stimulates pyridoxamine, whereas pentadecanoic acid (C15:0) does not show stimulation of pyridoxamine production.

Claims

CLAIMS1. The non-therapeutic use of one or more lipids to stimulate the mammalian gut microbiome of an individual to produce one or more vitamins comprising vitamin K or one or more B group vitamins.

2. Non-therapeutic use according to Claim 1 , wherein the use comprises in vivo stimulation of the mammalian gut microbiome; and wherein the use comprises administering a lipid composition comprising the one or more lipids to the individual.

3. Non-therapeutic use according to Claim 1 or Claim 2, wherein the individual is a human and wherein the mammalian gut microbiome is a human gut microbiome.

4. Non-therapeutic use according to any preceding claim, wherein the gut microbiome is stimulated to produce a greater amount of the one or more vitamins by the one or more lipids or lipid composition relative to the degree of stimulation by the same weight of glucose.

5. Non-therapeutic use according to any preceding claim, wherein the one or more lipids or the lipid composition comprises one or more free fatty acids or salts thereof; or one or more fatty acid esters such as one or more triglycerides, one or more diglycerides, one or more monoglycerides, one or more alkyl esters; or a combination thereof.

6. Non-therapeutic use according to any preceding claim, wherein the one or more lipids or lipid composition comprises one or more free fatty acids or salts thereof.

7. Non-therapeutic use according to any preceding claim, wherein the one or more lipids or lipid composition comprises at least 50% by weight of free fatty acids or salts thereof; preferably wherein the one or more lipids or lipid composition comprises at least 70% by weight of free fatty acids or salts thereof; and more preferably wherein the one or more lipids or lipid composition comprises at least 80% by weight of free fatty acids or salts thereof.

8. Non-therapeutic use according to any preceding claim, wherein the one or more lipids or lipid composition comprise free fatty acids having 18 carbon atoms or 12 carbon atoms, salts thereof, esters thereof such as triglyceride, diglyceride or monoglyceride esters thereof, or any combination thereof; preferably wherein the one or more lipids or lipid composition comprise lauric acid (C12:0), oleic acid (C18: 1), linoleic acid (C18:2), stearic acid (C18:0), salts thereof, esters thereof such as triglyceride, diglyceride or monoglyceride esters thereof, or any combination thereof.

9. Non-therapeutic use according to any preceding claim, wherein the one or more lipids or lipid composition comprise lauric acid (C12:0), oleic acid (C18: 1), linoleic acid (C18:2), salts thereof, esters thereof such as triglyceride, diglyceride or monoglyceride esters thereof, or a combination thereof.

10. Non-therapeutic use according to any preceding claim, wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises at least 40% by weight, and preferably at least 50% by weight, of a total of lauric acid (C12:0), oleic acid (C18:1 ) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.11 . Non-therapeutic use according to any preceding claim, wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises at least 60% by weight, and preferably at least 70% by weight, of a total of lauric acid (C12:0), oleic acid (C18:1 ) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

12. Non-therapeutic use according to any preceding claim, wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises at least 80% by weight, of a total of lauric acid (C12:0), oleic acid (C18:1 ) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition , free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

13. Non-therapeutic use according to any preceding claim, wherein the one or more lipids or lipid composition comprises pentadecanoic acid (C15:0) and / or heptadecanoicacid (C17:0), salts thereof, esters thereof such as triglyceride, diglyceride or monoglyceride esters thereof.

14. Non-therapeutic use according to any preceding claim, wherein the one or more vitamins comprise one or more B group vitamins; preferably, wherein the one or more B group vitamins comprise Vitamin Bi (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (Niacin), Vitamin Bs (pantothenic acid), Vitamin Be (pyridoxamine), Vitamin B? (biotin), Vitamin Bg (folic acid and folates), Vitamin B12 (cobalamines), or any combination thereof; and more preferably, wherein the one or more vitamins comprise Vitamin B2 (riboflavin), Vitamin Be (pyridoxamine), or any combination thereof.

15. Non-therapeutic use according to any preceding claim, wherein the one or more vitamins comprise riboflavin.

16. Non-therapeutic use according to Claim 15, wherein the one or more lipids or lipid composition comprises one or more free fatty acids having 12 carbon atoms, salts thereof or esters thereof such as triglyceride, diglyceride, monoglyceride, or alkyl esters thereof, or combinations thereof.

17. Non-therapeutic use according to Claim 15 or Claim 16, wherein the one or more lipids or lipid composition comprises lauric acid (C12:0), salts thereof, esters thereof such as triglyceride, diglyceride, monoglyceride, or alkyl esters thereof, or a combination thereof.

18. Non-therapeutic use according to any one of Claims 15 to 17, wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises at least 40% by weight, and preferably at least 50% by weight of lauric acid (C12:0), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition , free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

19. Non-therapeutic use according to any of Claims 15 to 18, wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises at least 60% by weight, and preferably at least 70% by weight, of lauric acid (C12:0), wherein said percentages of fattyacid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition , free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

20. Non-therapeutic use according to any of Claims 15 to 19, wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises at least 80% by weight, and preferably at least 90% by weight of lauric acid (C12:0, wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition , free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

21. Non-therapeutic use according to any of Claims 15 to 20, wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises, coconut oil, palm kernel oil, babassu oil, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, milk fat, any fraction thereof, any interesterified form thereof, or any combination thereof.

22. Non-therapeutic use according to any of Claims 15 to 21 , wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises free fatty acids derived from coconut oil, palm kernel oil, babassu oil, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, milk fat, any fraction thereof, or any combination thereof.

23. Non-therapeutic use according to any of Claims 1 to 14, wherein the one or more vitamins comprise pyridoxamine.

24. Non-therapeutic use according to Claim 23, wherein the one or more lipids or lipid composition comprises one or more free fatty acids having 18 carbon atoms, salts thereof or esters thereof such as triglyceride, diglyceride, monoglyceride, or alkyl esters thereof, or combinations thereof; preferably, wherein the one or more lipids or lipid composition comprises oleic acid (C18:1 ), linoleic acid (C18:2), stearic acid (C18:0), salts thereof oresters thereof such as triglyceride, diglyceride, monoglyceride or alkyl esters thereof, or combinations thereof.

25. Non-therapeutic use according to Claim 23 or Claim 24, wherein the one or more lipids or lipid composition comprises oleic acid (C18:1 ), linoleic acid (C18:2), salts thereof, esters thereof such as triglyceride, diglyceride, monoglyceride, or alkyl esters thereof or a combination thereof.

26. Non-therapeutic use according to any one of Claims 23 to 25, wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises at least 70% by weight of a total of oleic acid (C18:1 ) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

27. Non-therapeutic use according to any one of Claims 23 to 26, wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises at least 80% by weight of a total of oleic acid (C18:1 ) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

28. Non-therapeutic use according to any one of Claims 23 to 27, wherein the use comprises administering a lipid composition comprising the one or more lipids to an individual; and wherein the lipid composition comprises at least 90% by weight of a total of oleic acid (C18:1 ) and linoleic acid (C18:2), wherein said percentages of fatty acid residues refers to free fatty acids, fatty acids present in a salt form, and fatty acids bound as acyl groups in esters in the lipid composition and being based on the total weight of C4 to C24 fatty acid residues bound as acyl groups present in the lipid composition, free fatty acid residues, and fatty acid residues present in a salt form present in the lipid composition.

29. Non-therapeutic use according to any of Claims 23 to 28, wherein the lipid composition comprises soyabean oil, sunflower oil, rapeseed oil, groundnut oil, cottonseed oil, maize oil, safflower oil, sesame oil, rice bran oil, evening primrose oil, borage oil, palm oil, a microbially derived oil, any fraction thereof, any interesterified form thereof, or any combination thereof.

30. Non-therapeutic use according to any of Claims 23 to 29, wherein the lipid composition comprises free fatty acids derived from soyabean oil, sunflower oil, rapeseed oil, groundnut oil, cottonseed oil, maize oil, safflower oil, sesame oil, rice bran oil, evening primrose oil, borage oil, palm oil, a microbially derived oil, any fraction thereof, any interesterified form thereof, or any combination thereof; or wherein the lipid composition comprises free fatty acids derived from microbial fermentation.

31. Non-therapeutic use according to any of Claims 2 to 30, wherein the lipid composition comprises soyabean oil, sunflower oil, rapeseed oil, groundnut oil, cottonseed oil, maize oil, safflower oil, sesame oil, rice bran oil, evening primrose oil, borage oil, palm oil, a microbially derived oil, coconut oil, palm kernel oil, babassu oil, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, milk fat, any fraction thereof, any interesterified form thereof, or any combination thereof.

32. Non-therapeutic use according to any of Claims 2 to 31 , wherein the lipid composition comprises free fatty acids derived from soyabean oil, sunflower oil, rapeseed oil, groundnut oil, cottonseed oil, maize oil, safflower oil, sesame oil, rice bran oil, evening primrose oil, borage oil, palm oil, a microbially derived oil, coconut oil, palm kernel oil, babassu oil, cohune oil, murumuru oil, ouricury oil, tucum oil, genetically modified rapeseed oil, milk fat, any fraction thereof, or any combination thereof; orwherein the lipid composition comprises free fatty acids derived from microbial fermentation.

33. Non-therapeutic use according to any of Claims 2 to 32, wherein the lipid composition is formulated for colonic delivery of the one or more lipids.

34. Non-therapeutic use according to Claim 33, wherein the lipid composition is formulated for rectal administration and wherein the lipid composition is administered to the individual by rectal administration.

35. Non-therapeutic use according to Claim 34, wherein the lipid composition is formulated for oral delivery and wherein the lipid composition is administered to the individual by oral administration.

36. Non-therapeutic use according to Claim 33 or Claim 35, wherein the lipid composition is encapsulated with one or more of a pH sensitive polymer; a delayed release polymer; and a polymer that is degradable by one or more microbes of the mammalian gut microbiome.

37. Non-therapeutic use according to any of Claims 32, 34 or 35, wherein the lipid composition is coated with one or more of the following polymers: cellulose acetate phthalate; cellulose acetate succinate; cellulose acetate trimellitate; polyvinyl acetate phthalate; hydroxypropylmethylcellulose phthalate; hydroxypropylmethylcellulose acetate succinate; and a copolymer of two or more of methacrylic acid, acrylic acid, salts thereof and esters thereof.

38. Non-therapeutic use according to any of Claims 33 or 35 to 37, wherein the lipid composition is coated with one or more polysaccharides; optionally wherein the one or more polysaccharides comprise chitosan, pectin, starch, chondroitin and salts thereof, alginate, gums, amylose, dextran, inulin, galactomannan, cyclodextrins, cellulose, derivatives thereof, or combinations thereof.

39. Non-therapeutic use according to any one of Claims 33 or 35 to 38, wherein the lipid composition is present within a delayed release matrix.

40. Non-therapeutic use according to Claim 39, wherein the delayed release matrix is a polysaccharide matrix.

41. Non-therapeutic use according to Claim 39, wherein the polysaccharide matrix comprises chitosan, pectin, starch, chondroitin and salts thereof , alginate, gums, amylose, dextran, inulin, galactomannan, cyclodextrins, cellulose, derivatives thereof, or combinations thereof.

42. Non-therapeutic use according to any one of Claims 33 or 35 to 41 , wherein the lipid composition is encapsulated by oleosomes.

43. Non-therapeutic use according to any one of Claims 33 or 35 to 42, wherein the lipid composition comprises one or more lipids that comprise a lipid moiety covalently bound to a non-lipid moiety.

44. Non-therapeutic use according to Claim 43, wherein the lipid moiety comprises a fatty acid moiety; preferably, wherein the fatty acid moiety is lauric acid (C12:0), stearic acid (C18:0), oleic acid (C18:1 ) or linoleic acid (C18:2) moiety.

45. Non-therapeutic use according to Claim 43 or Claim 44, wherein the non-lipid moiety comprises a polysaccharide moiety; preferably wherein the polysaccharide moiety is a fiber moiety.

46. Non-therapeutic use according to any one or more of Claims 2 to 45, wherein the use comprises administration of a lipid composition comprising one or more lipids to the individual; and wherein the lipid composition comprises from 0.5 grams to 10 grams of the one or more lipids per serving.

47. Non-therapeutic use according to Claim 46, wherein the lipid composition comprises from 0.5 grams to 10 grams of a total of lauric acid (C12:0), oleic acid (C18:1 ), linoleic acid (C18:2), and salts and esters thereof per serving.

48. Non-therapeutic use according to Claim 46 or Claim 47, wherein the lipid composition is a unit dosage form comprising from 0.5 grams to 10 grams of the one or more lipids.

49. Non-therapeutic use according to Claim 48, wherein the lipid composition is a unit dosage form comprising from 0.5 grams to 10 grams of a total of lauric acid (C12:0), oleic acid (C18:1 ), linoleic acid (C18:2), and salts and esters thereof .

50. Non-therapeutic use according to any of Claims 46 to 49, wherein the one or more vitamins comprise riboflavin; and wherein the lipid composition comprises from 0.5 grams to 10 grams of a total of lauric acid (C12:0), and salts and esters thereof per serving.51 . Non-therapeutic use according to any of Claims 46 to 49, wherein the one or more amino acids comprise pyridoxamine; and wherein the lipid composition comprises from 0.5 grams to 10 grams of a total of oleic acid (C18:1 ) and linoleic acid (C18:2), and salts and esters thereof per serving.

52. Non-therapeutic use according to any preceding claim, wherein the use comprises administration of from 0.5 grams to 10 grams per day of the one or more lipids to the individual.

53. Non-therapeutic use according to any preceding claim, wherein the use comprises administration of from 0.5 grams to 10 grams per day of a total of lauric acid (C12:0), oleic acid (C18:1 ), linoleic acid (C18:2), and salts and esters thereof to the individual.

54. Non-therapeutic use according to any preceding claim, wherein the use comprises administration of from 0.5 grams to 10 grams per day of a total of lauric acid (C12:0), and salts and esters thereof to the individual.

55. Non-therapeutic use according to any preceding claim, wherein the use comprises administration of from 0.5 grams to 10 grams per day of a total of oleic acid (C18:1 ), linoleic acid (C18:2), and salts and esters thereof to the individual.

56. A nutraceutical product comprising a lipid composition comprising one or more lipids, wherein the lipid composition comprises lauric acid (C12:0), oleic acid (C18:1), linoleic acid (C18:2), salts thereof, esters thereof, or a combination thereof; wherein the lipid composition is formulated for colonic delivery of the one or more lipids.

57. A neutraceutical product according to Claim 56, wherein the one or more lipids or lipid composition comprises pentadecanoic acid (C15:0) and / or heptadecanoic acid (C17:0), salts thereof, esters thereof, or a combination thereof.

58. A nutraceutical product according to Claim 56 or 57, wherein the lipid composition is as defined in any one or more Claims 5 to 12, 15 to 21 , 23 to 32 and 34 to 44.

59. A nutraceutical product according to any one of Claims 56 to 58, wherein the lipid composition comprises lauric acid (C12:0), oleic acid (C18:1 ) and linoleic acid (C18:2).

60. A nutraceutical product according to any one of Claims 56 to 59, wherein the product comprises the lipid composition in an amount of from 0.5% to 100% by weight of the nutraceutical product.

61. A nutraceutical product according to any of Claims 56 to 60, wherein the nutraceutical product comprises from 0.5 grams to 10 grams of a total of lauric acid (C12:0), oleic acid (C18:1 ), linoleic acid (C18:2), and salts and esters thereof per serving.

62. A nutraceutical product according to any of Claims 56 to 61 , wherein the nutraceutical product comprises from 0.5 grams to 10 grams of lauric acid (C12:0), and salts and esters thereof per serving.

63. A nutraceutical product according to any of Claims 56 to 62, wherein the nutraceutical product comprises from 0.5 grams to 10 grams of a total of oleic acid (C18:1 ) and linoleic acid (C18:2), and salts and esters thereof per serving.

64. A nutraceutical product according to any one of Claims 56 to 63, wherein the product is in solid form; preferably wherein the product is a powder, tablet, dough, bar or capsule.

65. A nutraceutical product according to Claim 64, wherein the product comprises the lipid composition in an amount of from 5% to 100% by weight.

66. A nutraceutical product according to any one of Claims 56 to 60, wherein the nutraceutical product is a food product or an infant formula.

67. A nutraceutical product according to any one of Claims 56 to 65, wherein the nutraceutical product is a unit dosage form comprising from 0.5 grams to 10 grams of a total of lauric acid (C12:0), oleic acid (C18: 1 ), linoleic acid (C18:2), and salts and esters thereof.

68. A nutraceutical product according to any one of Claims 56 to 63, wherein the product is in liquid form; preferably wherein the product is a drink, yoghurt or cream.

69. A nutraceutical product according to Claim 68, wherein the product comprises the lipid composition in an amount of from 0.5% to 10% by weight.

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