Food composition comprising fatty acid amide (FAA)-producing bacteria

Abstract

The present invention relates to a method for providing a food composition, wherein the method comprises the step of fermenting a fermentable food substrate using a bacterium capable of producing providing a fatty acid amide (FAA) to obtain a fermented food substrate. Preferably, the bacterium capable of producing a fatty acid amide (FAA) is Coprococcus eutactus, or Coprococcus aceti.

Description

[0001] P36849PCOO / MJO

[0002] Title: Food composition comprising FAA-producing bacteria

[0003] Technical field

[0004] The present invention relates to a method for providing a fermented food using a bacterium capable of producing a fatty acid amid (FAA), a food composition obtained or obtainable by a method according to the disclosure, and the food composition according to the disclosure for use in the prevention or treatment of neurological disorders.

[0005] Background of the invention

[0006] In today's society, there is a notable increase in mental disorders, including depression and anxiety disorders, which are among the top ten leading causes of health- related burdens worldwide. Apart from the significant impact on quality of life, these disorders impose a heavy financial burden on society.

[0007] Gut bacteria play vital roles in nutrient digestion, metabolism, fermentation, and endocrine and immunomodulatory functions. Most gut bacteria reside in the large intestine (colon) and perform fermentation, while the remainder live in the stomach and small intestine. Fermentation refers to enzymatically breaking down carbohydrates, extracted from e.g. dietary fibers, into other compounds, for example releasing crucial metabolites called shortchain fatty acids (SCFAs), such as acetate, butyrate, and propionate, as well as amines, vitamins, gases, and alcohols. Though butyrate is the least abundant SCFA, it is the primary energy source for epithelial cells in the gut lumen barrier called colonocytes. Butyrate undergoes beta-oxidation in colonocytes, leading to the production of acetyl-CoA, which then enters the citric acid cycle (Krebs cycle) to generate ATP, the energy currency of cells.

[0008] The gut barrier’s integrity depends to a large extent on the cross-talk between gut microbiota and the colonocytes. The gut microbiota maintains gastrointestinal tract homeostasis and fends off pathogenic bacteria and pathobionts. To some extent dysbiosis results from a low level of butyrate-producing bacteria, leading to disproportionate production of SCFAs. This disrupts the cross-talk, and oxygen permeates the lumen’s hypoxic environment. More specifically, the disruption of the hypoxic environment in the gut allows more aerotolerant bacteria to thrive, which contributes to dysbiosis. Dysbiosis is concerning because it has been linked to cancer development, allergic airway diseases, autoimmune disorders, cardiovascular diseases, neurological disorders, metabolic diseases and other diseases.

[0009] Recent research has revealed a strong association between these disorders and the composition and activity of the gut microbiome. Specifically, depression and related symptoms like anxiety are linked to gut-brain interactions mediated by the vagus nerve, the peripheral immune system, as well as metabolites produced by the gut microbiota. A number of independent scientific studies conducted over the past decade, including large cohort studies with over 1 ,000 participants, have highlighted an inverse correlation of the gut bacterium Coprococcus eutactus with depression and anxiety. This gut bacterium has consistently shown to be depleted or very low abundance below detection levels in individuals with neuropsychological and neurodegenerative disorders, such as Parkinson’s Disease, schizophrenia, autism, anxiety, and depression. Conversely, high levels of Coprococcus have been positively correlated with cognitive development, motivation to exercise, and overall quality of life indicators.

[0010] Evidence from a progressively increasing number of studies suggests that Coprococcus eutactus is a crucial player in the gut-brain axis (GBA). This non-motile, ethanol-resistant (in the case of its spores), mesophilic, Gram-positive, obligately anaerobic gut bacterium is highly abundant in the colon. Coprococcus eutactus actively ferments dietary fibers, especially carbohydrates such as p-glucans, and can convert these into butyric acid, acetic acid, formic acid, and lactic acid. This butyrate-producing species has received heightened attention because it was correlated with health versus disease status. Coprococcus eutactus was observed to be depleted in patients with neurological disorders, such as Parkinson’s Disease, autism, depression, and schizophrenia, and metabolic disorders, such as inflammatory bowel disease (IBD) and preeclampsia during pregnancy. Conversely, Coprococcus eutactus was positively correlated with quality of life indicators and elevated levels of the species appeared in Ugandan children with higher language development.

[0011] A leading hypothesis elucidates the indirect involvement of Coprococcus eutactus in the neuronal, metabolic, and immune processes of the GBA. Within the Class Clostridia, conservation of biosynthetic gene clusters (BGCs) was identified. These BGCs are presumed to contain enzymes that conjugate fatty acids (FAs) with neurotransmitter amines to produce fatty acid amides (FAAs). It is assumed that the two substrates, FA and amines, originate from the host's cells, diet, or other gut bacteria and that the FAAs created structurally mimic the endogenous FAAs of the host. Through this mimicry, the FAAs produced by Coprococcus eutactus act as agonists that interact with host G-protein coupled receptors (GPCRs) involved in the pathways of the nervous system, thereby influencing the host's mood and behavior.

[0012] This inferred mechanism may also help explain why patients with reduced levels of Coprococcus eutactus in their gut often exhibit neurological disorders such as Parkinson's Disease, schizophrenia, autism, anxiety, and depression . The low levels of specific FAAs might result from the decreased abundance of C. eutactus, potentially contributing to the development or exacerbation of these neurological conditions. Dysbiosis in the colon is linked to dysfunction of the nervous system. The presence of few butyrate-producing microbes affects the epithelial barrier function, allowing different types of bacteria and / or their metabolites and / or other components present in the Gl tract to diffuse through the gut lumen, resulting in inflammation, reinforcing dysbiosis and reducing production of essential metabolites such as SCFA and other key metabolites.

[0013] A growing body of publications reports on the common finding that neurological disorders and colon dysbiosis are correlated, and their findings corroborate the theory that the gut influences brain function and mental health. In one recent rodent study, FAA- supplemented diets enhanced motivation to exercise by augmenting dopamine signaling via the dorsal root ganglia. It was demonstrated that the FAAs produced by exercise-enhancing gut bacteria peripherally activated the endocannabinoid receptors (CB1) expressed by TRPV1 -expressing sensory neurons. FAAs interacted as structural mimics of the natural ligands to such receptors, the effect of which elevated dopamine levels in exercising mice. FAAs triggered peripheral CB1 signaling, inhibiting dopamine degradation (by monoamine oxidase (MAO)), an action not demonstrated in microbiome-depleted mice. Taking it further by colonizing germ-free mice with Escherichia coli endowed with the FAA-producing BGC increased exercise capacity and dopamine signaling. A second rodent experiment that linked Coprococcus eutactus as a modulator of brain activity illustrated that transplantation of Coprococcus eutactus significantly attenuated chronic restraint stress (CRS)- induced depressionin mice i and prevent synaptic loss and glial neuroinflammation.

[0014] Despite the promising potential of Coprococcus eutactus and its metabolites on mood and behavior, there are currently no commercially available probiotics, functional foods, supplements or medicaments that are based on this bacterium. The primary challenge lies in the technical difficulties associated with culturing Coprococcus eutactus at a commercial scale. This bacterium is anaerobic and highly sensitive to oxygen. These factors collectively hinder the development of Coprococcus eutactus -based products that can be reliably produced, stored, and administered to consumers.

[0015] Moreover, while Coprococcus eutactus shows significant promise due to its butyrate- producing capabilities and positive correlations with mental health, there is a lack of specific methods for providing this bacterium in a viable form as a probiotic. The complexity of the gut microbiome and the interactions between different bacterial species further complicate the development of targeted interventions.

[0016] Hence, there is a need to develop effective, science-based probiotics and supplements.

[0017] Considering the above, it is an object of the present invention amongst other objects, to provide effective, science-based probiotics, probiotic fermented food, probiotics containing food and probiotics containing supplements or medicaments. It is an object of the present invention, amongst other objects, to meet the above unmet need in the art.

[0018] Summary of the invention

[0019] The present invention meets the above object, amongst other objects, as outlined in the appended claims.

[0020] Specifically, the present disclosure meets one or more of the above-mentioned objectives by providing a method for providing a food composition, wherein the method comprises a step of fermenting a fermentable food substrate using a bacterium capable of providing a fatty acid amide (FAA), preferably Coprococcus spp., more preferably Coprococcus eutactus, to obtain a fermented food product.

[0021] The present invention also relates to a food composition obtainable or obtained by a method according to the present disclosure.

[0022] The present invention, according to another aspect, relates to a food composition comprising a fatty acid amide (FAA) and an FAA-producing bacteria, preferably Coprococcus spp., more preferably Coprococcus eutactus.

[0023] The present invention also encompasses the use of a bacterium capable of producing a fatty acid amide (FAA), preferably Coprococcus spp., more preferably Coprococcus eutactus, for fermenting a fermentable food substrate.

[0024] An advantage of the food composition according to the present disclosure and the method for providing such a food composition is that it provides a food composition with an immediate impact that provides a more immediate effect, along with a gradual, sustained effect that ensures long-term effectiveness.

[0025] Brief description figures

[0026] Figure 1. The decrease in pH resulting from overnight fermentation by Coprococcus eutactus ATCC 27759 in oat-based media. The two population groups (t=0 and t=24) have means that are significantly different from each other. T-tests comparing t=0 with t=24 per oat media were performed. (**** means p-value < 0.0001).

[0027] Figure 2. Coprococcus eutactus density counted as CFU / ml as a result of overnight incubation at 37 °C in replicate measurements in oats-based media (n=3) compared to the regular growth media, Wilkins-Chalgren (WC) Anaerobe broth (n=2). Error bars indicate the standard deviation. CFU, colony forming unit. One t-test was performed per oat media. (* means p-value < 0.05, *** means p-value < 0.001, **** means p-value < 0.0001) Detailed description of the invention

[0028] In a first aspect, the present disclosure provides a method for providing a food composition, wherein the method comprises the step of fermenting a fermentable food substrate using a bacterium capable of providing a fatty acid amide (FAA) to obtain a fermented food composition.

[0029] The indefinite article “a” or “an” can be used interchangeably with the phrases “one or more” or “at least one”. As such, the articles “a” and “an” should be understood to mean one, two, three, four, five, six, or more.

[0030] Fermentation refers to the bacterial breakdown of the fermentable food substrate, leading to the provision of a fatty acid amide (FAA) and optionally further compounds such as short-chain fatty acids, bioactive peptides, or other metabolites. In other words, in the present disclosure, the bacterium can provide said at least one FAA in the fermenting process.

[0031] The advantage of fermenting a food substrate with a health beneficial bacterium capable of providing a fatty acid amide (FAA) and / or other health functional metabolites is the provision of a food product containing the probiotic strain and / or its metabolites with an improved health functionality. The fermentation conditions place the probiotic bacteria in a more optimal metabolic state, allowing them to adapt to the substrate. This is believed to enhance their ability to colonize the gut after being consumed.

[0032] In one embodiment, the fermentable food substrate is fermented at a temperature optimized for the growth and metabolic activity of the selected bacterial strain(s). The temperature range for the fermentation process is preferably between 20°C and 45°C. For example, when using Coprococcus eutactus, the preferred fermentation temperature is between 30°C and 41 °C. This temperature range ensures optimal bacterial activity and efficient fermentation of the food substrate, resulting in the production of beneficial metabolites such as short-chain fatty acids, bioactive peptides, and other compounds. The temperature may be maintained using a controlled fermentation apparatus equipped with temperature regulation capabilities to ensure consistent and reproducible results.

[0033] In one embodiment, the fermentable food substrate is provided in a growth medium. The growth medium supports growth and metabolic activity of the selected bacterial strain(s). The growth medium comprises a fermentable food substrate and optionally further protein, trace elements, vitamins, growth factors, and a buffering agent. Additionally or alternatively, the fermentable food substrate may itself further comprise protein, trace elements, and vitamins to enhance bacterial growth and metabolic activity. The protein may be a protein hydrolysate, such as yeast extract or a plant-derived protein hydrolysate. The trace elements may include, but are not limited to, calcium (Ca), zinc (Zn), cobalt (Co), manganese (Mn), iron (Fe), copper (Cu), and magnesium (Mg). Examples of vitamins are biotin, riboflavin, and niacin. A suitable buffering agent is a phosphate buffer, a citrate buffer, a Tris (tris(hydroxymethyl)aminomethane) buffer, an acetate buffer or succinate buffer. The concentration of the buffering agent(s) should be sufficient to maintain the pH of the growth medium within a pH of between 4.0 and 7.5, preferably 5.5 to 6.5. A skilled person will know which buffering agent to use and in which quantity to obtain the desired pH range.

[0034] In one embodiment, the fermentable food substrate or growth medium comprising the fermentable food substrate has a pH maintained within a range that supports the optimal growth and metabolic activity of the selected bacterial strain(s). The pH is preferably between 4.0 and 7.5. For instance, when fermenting with Coprococcus eutactus, the pH is preferably between 5.5 and 6.5. The pH can be adjusted and controlled using buffering agents or by periodic addition of acid or base as required. Maintaining the appropriate pH is crucial for maximizing the production of fatty acid amides (FAA) and ensuring the stability and viability of the bacterial culture throughout the fermentation process.

[0035] In one embodiment, the fermentable food substrate is fermented for a period of time sufficient for the bacterial strain(s) to metabolize the food substrate and produce fatty acid amides (FAA). The fermentable food substrate is preferably fermented for 12 hours to 96 hours, depending on the specific bacterial strain and the characteristics of the food substrate. For example, when using Coprococcus eutactus, the fermentable food substrate is preferably fermented for 24 to 72 hours. The duration is selected to ensure complete or partial fermentation of the substrate, resulting in the desired or optimal production of fatty acid amides, such as oleoyl aminovaleric acid and N-oleoylethanolamide (OEA). The progress of fermentation may be monitored by measuring parameters such as pH, substrate consumption, and metabolite production to determine the optimal endpoint.

[0036] In one embodiment, the fermentable food substrate is fermented under anaerobic conditions meaning oxygen is excluded or significantly limited, making it unsuitable for aerobic organisms that require oxygen for survival and metabolism, and / or enable anaerobic microorganisms to ferment the food substrate. Specifically, anaerobic conditions may be defined as conditions wherein a strictly anaerobic bacterium, such as Coprococcus spp. can survive, multiply and be metabolically active. In addition or alternatively, anaerobic conditions may be defined by an oxygen concentration (e.g. in the headspace of the fermentation vessel) of less than 1% (v / v), preferably less than 0.1% (v / v), and most preferably less than 0.01% (v / v). Additionally or alternatively, anaerobic conditions may be defined as the presence of a dissolved oxygen (DO) concentration in the growth medium of less than 0.1 parts per million (ppm), preferably less than 0.05 ppm, and most preferably less than 0.01 ppm. For example, when using Coprococcus eutactus, an anaerobic environment is used for the growth and Coprococcus eutactus.

[0037] The method for providing a food composition according to the present disclosure is (entirely) performed ex vivo, i.e. , outside of a living organism such as an animal or human subject. Fermenting the fermentable food substrate ex-vivo ensures that the fermentation occurs under fundamentally distinct from any in vivo processes that may occur within a living organism's digestive system. Unlike in vivo digestion, where food undergoes a series of enzymatic and mechanical breakdowns before reaching the gut microbiota, the ex-vivo fermentation allows for the fermentation of a pre-selected, potentially pre-processed food substrate. This key difference in the starting material, optionally combined with the precisely controlled environmental conditions, enables the production of a fermented food, and / or specific bacterial populations, fatty acid amide (FAA) and optionally further metabolites. The ex vivo fermentation thus provides a fermented food substrate with unique advantages in terms of consistency, scalability, and the potential for targeted probiotic and postbiotic production.

[0038] The ex-vivo fermenting of the fermentable food substrate may be performed in a bioreactor or fermentation apparatus. The skilled person will be aware of other methods for culturing bacteria besides bioreactors, including but not limited to shake flasks, static cultures, roller bottles, spinner flasks, microplate cultures, agar plates, continuous culture systems, airlift fermenters, membrane bioreactors, and perfusion culture systems. The selection of an appropriate culture method would depend on factors such as the bacterial strain, required culture volume, aeration needs, and the specific objectives of the fermentation process.

[0039] Preferably, fermenting the fermentable food substrate is performed in a bioreactor or fermentation apparatus. A bioreactor or fermentation apparatus allows for control of fermentation conditions, including temperature, pH, and oxygen levels, resulting in consistent high-quality bacterial populations and fatty acid amide (FAA) production. This ex vivo method ensures that the fermentation occurs under defined and reproducible conditions, distinct from any in vivo processes that may occur within a living organism's digestive system.

[0040] Bacterium capable of providing a fatty acid amide (FAA)

[0041] The bacterium capable of providing a fatty acid amide (FAA) may be selected based on its ability to provide a fatty acid amide (FAA) and / or metabolize a food substrate.

[0042] The terms “bacterium capable of providing a fatty acid amide (FAA)” and “FAA- producing bacterium” have, in the context of this disclosure, the same meaning and may be used interchangeably.

[0043] In one embodiment, the bacterium capable of providing a fatty acid amide (FAA) comprises a first polypeptide, a second polypeptide and / or a third polypeptide, wherein the first polypeptide comprises an amino acid sequence with at least 70, 80, 90%, preferably 95%, more preferably 98%, even more preferably 99%, most preferably 100% sequence similarity to SEQ ID No. 7; the second polypeptide comprises an amino acid sequence with at least 70, 80, 90%%, preferably 95%, more preferably 98%, even more preferably 99%, most preferably 100% sequence similarity to SEQ ID No. 8; and the third polypeptide comprises an amino acid sequence with at least 70, 80, 90%%, preferably 95%, more preferably 98%, even more preferably 99%, most preferably 100% sequence similarity to SEQ ID No: 9.

[0044] In one embodiment, the bacterium capable of producing a fatty acid amide (FAA) comprises a first nucleic acid (e.g. gene), a second nucleic acid (e.g. gene) and / or a third nucleic acid (e.g. gene), wherein the first nucleic acid encodes for a first polypeptide comprising an amino acid sequence with at least 70, 80, 90% preferably 95%, more preferably 98%, even more preferably 99%, most preferably 100% sequence similarity to SEQ ID No. 7; the second nucleic acid encodes for a second polypeptide comprising an amino acid sequence with at least 70, 80, 90% preferably 95%, more preferably 98%, even more preferably 99%, most preferably 100% sequence similarity to SEQ ID No 8; and the third nucleic acid encodes for a third polypeptide comprising an amino acid sequence with at least 70, 80, 90% preferably 95%, more preferably 98%, even more preferably 99%, most preferably 100% sequence similarity to SEQ ID No. 9.

[0045] A gene refers to a (segment of) nucleic acid encoding a protein or at least comprising a start codon and a stop codon. A gene preferably comprises all features required to provide a particular polypeptide under suitable conditions in a bacterial cell. Such features may include a promotor, a terminator, a coding sequence, an open reading frame, untranslated regions and / or further regulatory elements. A gene is capable of providing a polypeptide. A gene may provide a particular polypeptide under suitable conditions in a bacterial cell.

[0046] In one embodiment, the bacterium capable of producing a fatty acid amide (FAA) comprises a first polypeptide, a second polypeptide and a third polypeptide, wherein the first polypeptide comprises an amino acid sequence represented by SEQ ID No. 7; the second polypeptide comprises an amino acid sequence represented by SEQ ID No. 8; and the third polypeptide comprises an amino acid sequence represented by SEQ ID No. 9.

[0047] In one embodiment, the bacterium capable of producing a fatty acid amide (FAA) comprises a first gene, a second gene and a third gene, wherein the first gene encodes for a first polypeptide comprising an amino acid sequence represented by SEQ ID No. 7; the second gene encodes for a second polypeptide comprising an amino acid sequence represented by SEQ ID No. 8; and the third gene encodes for a third polypeptide comprising an amino acid sequence represented by SEQ ID No. 9.

[0048] In a preferred embodiment, the bacterium capable of providing a fatty acid amide (FAA) comprises a first gene, a second gene and / or a third gene, wherein the first gene comprises a nucleic acid sequence with at least 70, 80, 90%, preferably 95%, more preferably 98%, even more preferably 99%, most preferably 100% sequence similarity to SEQ ID No. 1; the second gene comprises a nucleic acid sequence with at least 70, 80, 90%%, preferably 95%, more preferably 98%, even more preferably 99%, most preferably 100% sequence similarity to SEQ ID No. 2; and the third gene comprises a nucleic acid sequence with at least 70, 80, 90%, preferably 95%, more preferably 98%, even more preferably 99%, most preferably 100% sequence similarity to SEQ ID No: 3. In particular a high sequence identity with SEQ ID NO:1 is preferred (e.g. in order to produce specifically oleoyl aminovaleric acid and / or N-oleoylethanolamide (OEA).

[0049] In one embodiment, the bacterium capable of producing a fatty acid amide (FAA) comprises a first gene, a second gene and a third gene, wherein the first gene comprises a nucleic acid sequence represented by SEQ ID No. 1 ; the second gene comprises a nucleic acid sequence represented by SEQ ID No. 2; and the third gene comprises a nucleic acid sequence represented by SEQ ID No. 3.

[0050] In one embodiment, the bacterium capable of producing a fatty acid amide (FAA) as described above is a genetically engineered bacterial strain. A genetically engineered bacterium" as used herein refers to a genetically modified bacterium or bacterial population that comprises one or more gene sequences (e.g. nucleic acid sequences) or activities distinct from a nucleic acid sequence or activity present in the bacteria prior to said genetic modification (e.g., host bacteria and / or wild type counterpart). In certain embodiments, an engineered bacteria includes a heterologous polynucleotide.

[0051] In one embodiment, the bacterium capable of producing a fatty acid amide (FAA) as described above the bacterium comprises a plasmid comprising at least the first gene as described above, the second gene as described above and / or the third gene as described above. Preferably, the first gene, the first gene as described above, the second gene as described above, and the third gene as described above are each present on one or more plasmids.

[0052] In one embodiment, the bacterium capable of producing a fatty acid amide (FAA) is a bacterium from a genus selected from the group consisting of Blautia, Clostridiales, Clostridium, Coprococcus, Eubacterium, Lachnoclostridium, Lachnospiraceae, Marvinbryantia, Ruminococcaceae, and Ruminococcus. Preferably, the bacterium capable of producing a fatty acid amide (FAA) is a bacterium selected from the group consisting of Blautia producta, Blautia sp. An81, Blautia sp. Marseille-P2398, Blautia wexlerae, Clostridiales sp., Clostridium celatum, Clostridium sp. CAG:413, Clostridium sp. L2-50, Clostridium sp. CAG.253, Coprococcus eutactus, Coprococcus hominis, Coprococcus aceti Eubacterium rectale, Lachnoclostridium clostridioforme, Lachnospiraceae str. KH1P17, Lachnospiraceae str. LC2019, Marvinbryantia formatexigens, Ruminococcaceae bacterium, Ruminococcus albus, and Ruminococcus sp. CAG.488. All these bacteria could be used for fermenting a fermentable food substrate. Whereas existing probiotics, primarily featuring Lactobacilli or Bifidus strains, often fail to colonize the gut if these types of bacteria are already present, the supplementation of the bacteria capable of providing a fatty acid amide as disclosed herein may have a high change of successful colonization, in particular Coprococcus eutactus which presence is known to be negatively correlate with neurological disorders. Particularly, in case a specific type of bacterium is absent in the host, the likelihood for colonisation is highly increased.

[0053] In one embodiment, the bacterium capable of producing a fatty acid amide (FAA) is selected from the group consisting of Coprococcus eutactus', Coprococcus hominis (DSM112732) and Coprococcus aceti. Preferably, the bacterium is Coprococcus eutactus.

[0054] In one embodiment, the bacterium capable of providing a fatty acid amide (FAA) is capable of providing a fatty acid amide (FAA) selected from the group consisting of oleoyl dopamine; oleoyl tyramine, oleoyl aminovaleric acid, a-linolenoyl phenylethylamine, caproyl tryptamine and lauroyl tryptamine; preferably oleoyl aminovaleric acid.

[0055] In preferred embodiments of the method according to the present disclosure, the bacterium capable of producing a fatty acid amide (FAA) is a Coprococcus eutactus bacterium capable of producing oleoyl aminovaleric acid.

[0056] The Coprococcus eutactus may be Coprococcus eutactus ATCC 27759. The genomic DNA sequence of Coprococcus eutactus ATCC 27759 is available at the ATCC Genome portal (accession: NZ_CP102278.1; accessed on July 26, 2024; Genbank Release 260: April 15, 2024). Alternatively, Coprococcus eutactus may be isolated from human feces. Coprococcus species, including C. eutactus, C. catus, and C. comes, were first isolated from human feces using strict anaerobic techniques (Holdeman LV, Moore WE. New genus, Coprococcus, twelve new species, and emended descriptions of four previously described species of bacteria from human feces. Int. J. Syst. Bacteriol. 24: 260-277, 1974).

[0057] Coprococcus eutactus referred to herein is a well-known bacterial species, and preferably has a 16S rRNA gene sequence with at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID No. 4 or the 16S rRNA gene sequence of the type strain of Coprococcus eutactus, deposited as ATCC 27759 (American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, United States of America).

[0058] The term “sequence identity” or “sequence similarity” refers to the percentage of nucleotides or amino acids that are identical between two sequences after they have been properly aligned. “Sequence identity” or “sequence similarity” may be determined by alignment of two polypeptides or two nucleotide sequences using global or local alignment algorithms. A person skilled in the art can use various sequence alignment tools, such as BLAST, to align both nucleotide and protein sequences. The percentage of “sequence identity” or “sequence similarity” is calculated by comparing the sequences over the length of the shorter sequence being assessed. It is further understood that, when referring to “sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids) are referred to.

[0059] In one embodiment, the bacterium capable of providing a fatty acid amide (FAA) is a bacterial composition comprising a first bacterium capable of providing a fatty acid amide (FAA) as disclosed above and a second bacterium, wherein the first and second bacterium are from a different bacterial strain or species. The second bacterium may be selected from the group consisting of Lactobacillus spp., Bifidobacterium spp., Eubacterium spp., Streptococcus spp., Pediococcus spp., Clostridium spp., Weissella ssp., and a bacterium, capable of providing a fatty acid amide (FAA) as described above. Preferably, the second bacterium is selected from the group consisting of Streptococcus spp, Lactobacillus spp., Bifidobacterium spp., and a bacterium, capable of providing a fatty acid amide (FAA) as described above. If the second bacterium is an Eubacterium spp. bacterium, the second bacterium is preferably selected from the group consisting of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus salivarius. If the second bacterium is a Bifidobacterium spp., the second bacterium is preferably selected from the group consisting of Bifidobacterium bifidum, Bifidobacterium lactis, and Bifidobacterium longum. If the second bacterium is a Streptococcus spp. bacterium, the second bacterium is preferably Streptococcus thermophilus. If the second bacterium is a Eubacterium spp. bacterium, the second bacterium is preferably Eubacterium rectale. If the second bacterium is a Pediococcus spp. bacterium, the second bacterium is preferably Pediococcus pentosaceus. The bacterial composition may comprise at least one, at least two, at least three, at least four, at least five, or more bacteria as disclosed above, wherein each bacterium is from a different bacterial strain or species.

[0060] The one or more bacterium according to the present disclosure may (in part) be viable, attenuated, dead, or in form of spores.

[0061] In one embodiment, the bacterium capable of providing a fatty acid amide (FAA) is combined with the fermentable food substrate prior to fermenting the fermentable food substrate. Preferably said bacterium / inoculum is combined (or comprised) in an amount of 10A2.5 to 10A10 or 10A2.5 to 10A8 or to 10A7.4 CFU / ml or CFU / g, more preferably 10A3.5 to 10A6.4 CFU / ml or CFU / g, more preferably 10A4.5 to 10A5.4 CFU / ml or CFU / g, most preferably 10A4-10A7 CFU / ml or CFU / g.

[0062] The term "CFU" stands for "colony-forming unit." This is a measure used to estimate the number of viable bacteria in a sample. The CFU count is determined by culturing the microorganisms on a nutrient agar plate and counting the number of colonies that form. Each colony arises from a single microorganism or a group of microorganisms that were present in the sample, indicating the number of viable cells capable of growth and division. Fermentable food substrate

[0063] In one embodiment, the fermentable food substrate comprises a compound selected from the group consisting of a monosaccharide, a disaccharide, and a polysaccharide. The monosaccharide is preferably selected from the group consisting of glucose, fructose, mannose, arabinose, xylose, and rhamnose. The disaccharide is preferably selected from the group consisting of sucrose, lactose, maltose and cellobiose. The polysaccharide may be an oligosaccharide. The polysaccharide is preferably selected from the group consisting of raffinose, starch, cellulose, p-glucan (beta-glucan), hemi-cellulose, lichenan, pectin, a hydrocolloid and a gum. The starch may be potato-starch. The hemi-cellulose is preferably xylan or arabinoxylan. The hydrocolloid or gum is preferably selected from the group consisting of glucomannan, galactomannan and galactan. The food substrate may comprise one or more compounds as disclosed above.

[0064] In addition or alternatively, the fermentable food substrate may comprise any (C. eutactus fermentable) compound as disclosed in Table 2 of Holdeman LV, Moore WE. Int. J. Syst. Bacteriol. 24: 260-277, 1974. Or for example pectin, gum acacia, inulin, resistant starch and other carbohydrates indigestible to the human body and comprised of backbone of single carbohydrates or a combination of one or more of glucose, rhamnose, galalactose, xylose, arabinose, fructose, mannose.

[0065] In one embodiment, the fermentable food substrate comprises a compound selected from the group consisting of glucose, fructose, mannose, cellobiose, p-glucan, lichenan, glucomannan, galactan, pectin, and starch. Such compounds are particularly preferred if the bacterium capable of providing a fatty acid amide is a Coprococcus sp. bacterium, preferably a Coprococcus eutactus bacterium.

[0066] In one embodiment, the fermentable food substrate comprises a beta-glucan. Betaglucans are a group of polysaccharides composed of D-glucose monomers linked by beta- glycosidic bonds. They are found in the cell walls of cereals (like oats and barley), fungi, yeast, and some bacteria. Beta-glucans promote digestive health by increasing stool bulk and regularity. They may also act as prebiotics, promoting the growth of beneficial gut bacteria.

[0067] It was found that when using a fermentable food substrate comprising beta-glucan, a food composition was obtained with further utility, in particular enhanced probiotic properties, e.g. health benefits. The selection of a food composition comprising a beta-glucan derived from cereals was found to be particularly advantageous, especially when the bacterium capable of providing a fatty acid amide (FAA) is Coprococcus eutactus.

[0068] In one embodiment, the fermentable food substrate comprises a food selected from the group consisting of cereal, dairy, legume, fruit, and vegetable; or any combination of these foods. If the food is cereal, the cereal is preferably selected from the group consisting of oats, barley, rice wheat, rye, and sorghum. If the food is a fruit, the fruit may be selected from the group consisting of apple, pear, and banana. If the food is a legume, the legume may be selected from the group consisting of beans, lentils, and peas. If the food is dairy, the dairy is preferably selected from the group consisting of milk, yoghurt, buttermilk, kefir and whey. The fermentable food substrate may comprise one or more foods from any of these groups, either individually or in combination.

[0069] In one embodiment, the fermentable food substrate comprises a cereal selected from the group consisting of oats, barley, wheat and rye. Preferably, the cereal is oats or barley as these provide an improved fermentable food substrate compared to wheat and rye, in particular for Coprococcus eutactus. More preferably, the cereal is oats.

[0070] An advantage of a fermentable food substrate comprising oats is that oats were found to provide an improved fermentable substrate for Coprococcus eutactus, while allowing the provision of a food gluten-free food composition that may be in a form varying from beverages to baked goods.

[0071] In a preferred embodiments, the fermentable food substrate is a composition comprising a cereal as disclosed above and further comprising dairy. Preferably, the fermentable food substrate comprises cereals, preferably oats; more preferably oats and dairy; even more preferably oats, dairy and glucose. Dairy products, which contain lactose and other nutrients, further enhanced the fermentation process, in particular the combination was shown to provide a conducive environment for the growth of Coprococcus eutactus. The combination of cereals, in particular oats and barley, in combination with dairy provide an improved production of fatty acid amide (FAA) compared to other fermentable food substrates. Preferably, the composition further comprises glucose.

[0072] In one embodiment, the fermentable food substrate comprises 2.5% to 20% (w / v) oats, preferably 5% to 15% (w / v), more preferably 7.5% to 12.5% (w / v) oats.

[0073] In one embodiment, the fermentable food substrate comprises 1 to 99 wt.% of a dairy product, preferably milk. Preferably the food substrate comprises 50 to 95 wt.% of a dairy product, preferably milk.

[0074] In one embodiment, the fermentable food substrate comprises 0.1% to 5% (w / v) of a monosaccharose, preferably 0.2% to 2.5% (w / v) of a monosaccharose, more preferably 0.1% to 5% (w / v) of a monosaccharose. Preferably the monosaccharose is glucose. In addition or alternatively, additional fermentable carbohydrates (not naturally present) are added to the food substrate (enrichment of the food substrate), such as cellobiose.

[0075] In one embodiment, the method for providing a food composition according to the present disclosure comprises the step of fermenting a fermentable food substrate using Coprococcus eutactus to obtain a fermented substrate, wherein the fermentable food substrate is a composition comprising oats and preferably dairy. The dairy may be selected from the group consisting of milk, yoghurt, buttermilk, kefir and whey.

[0076] In one embodiment, the fermentable food substrate is pre-treated prior to fermenting. Pre-treatment of the fermentable food substrate may comprise one or more treatments selected from the group consisting of milling, soaking, cooking, enzymatic treatment, a pH adjustment, dialysis, sterilization and a concentration adjustment. Preferably the food substrate is sterilized, preferably by heating, to ensure that the fermentable food substrate is solely performed using a bacterium capable of producing a fatty acid amide (FAA) as defined above or a bacterial composition thereof as defined above. Enzymatic treatment may comprise treatment with alpha-amylase, pancreatin and / or pepsin and / or other carbohydrases or proteases or lipases. Preferably, the concentration of the fermentable food substrate is combined with a growth medium and its concentration adjusted so that the fermentable food substrate is present at 5-10 g / L in the growth medium. Pre-treatment of the food substrate may enhance fermentability (e.g., milling, soaking, or cooking).

[0077] In one embodiment, the food composition comprising the fermented food substrate is processed after fermentation. Processing may comprise drying, blending, or packaging, to create the final food composition.

[0078] Sporulation

[0079] In one embodiment, a method for providing a food composition as disclosed above comprises the further step of inducing the bacterium to sporulate, preferably after fermenting the fermentable food substrate.

[0080] An advantage of inducing sporulation at the end the fermentation process, or after the fermentation has been completed, is that the amount of fatty acid amides (FAA) is optimized. If sporulation were induced earlier, the amount of these fatty acid amides (FAA) may be limited or reduced. Furthermore, allowing the fermentation process to complete before inducing sporulation results in a higher number of bacteria that can form spores, increasing the probiotic potency of the final product.

[0081] Sporulation is a survival mechanism employed by certain bacteria, allowing them to withstand unfavorable environmental conditions. Inducing sporulation can provide several advantages for the food composition. Spores are highly resistant to environmental stressors, such as heat, desiccation, and UV radiation, enhancing the shelf stability of the product. This resistance ensures that following sporulation the fermenting bacteria remain in viable form during storage and transportation. Additionally, spores can survive the acidic environment of the stomach, allowing them to reach the intestines where they can germinate and exert their probiotic effects. This targeted delivery enhances the efficacy of the probiotic bacteria. In particular, sporulation allows for the development of novel fermented products, such as a baked spore-comprising food composition, whereby the spores can germinate and whereby the spores originate from health beneficial microbes.

[0082] Sporulation may be induced through various means, including but not limited to nutrient depletion, exposure to oxygen, pH changes, temperature shifts, preferably heat, and the addition of specific sporulation-inducing compounds, e.g., alcohols such as ethanol. Nutrient depletion involves limiting the availability of essential nutrients, triggering the bacteria to form spores. Exposure to oxygen can induce sporulation in anaerobic bacteria like Coprococcus spp., pH changes, such as acidification, can also trigger sporulation. Temperature shifts, such as heating or cooling, can promote spore formation. Additionally, specific compounds known to induce sporulation can be added to the fermentation medium. These methods can be optimized based on the specific bacterial strain and the characteristics of the food substrate.

[0083] Nutrient depletion may be used to induce sporulation after fermentation. An advantage of nutrient depletion-induced sporulation is that it is believed to create a more effective food product. Nutrient depletion refers to a state where the nutrients required for microbial growth and metabolism have been reduced or exhausted in the growth medium or substrate. This condition typically occurs towards the end of the fermentation process when the microorganisms have consumed most of the available nutrients. In particular, nutrient depletion refers to a state where the growth medium or substrate, in order of preference, comprises less than 0.1% (w / v), 0.08% (w / v), 0.05% (w / v), or 0.02% (w / v) of a fermentable food substrate, e.g., glucose.

[0084] In one embodiment, the method for providing a food composition according to the present disclosure may further comprise a step to induce sporulation. Verification may be performed through microscopic examination, where the presence of spores can be visually confirmed. Viability testing under stress conditions, such as oxygen or acid exposure, can also be conducted to assess the presence and resistance of the spores. Additionally, molecular techniques, such as PCR and flow cytometry, can be used to quantify the number of spores and confirm their identity, following or not following a germination. The further confirmation of sporulation step ensures that sporulation has been successfully induced and that the spores are viable and capable of germination.

[0085] After sporulation, the food composition may undergo further processing steps to ensure the stability and efficacy of the food composition. These steps may include drying or lyophilization to create a powder form, which can be easily incorporated into various food products. The spores can also be incorporated into different food matrices, such as beverages, snacks, and supplements, to create a wide range of products. The spores can also be incorporated in food composition that can serve as a medicine. These processing steps ensure that the spores remain viable and effective until consumption. It is also foreseen that the spores (e.g. isolated / purified from the food composition) are used separately as a supplement or medicine. Accordingly, the present disclosure also provides for a composition, e.g. a supplement or medicine, comprising spores as taught herein, for example in amounts as taught herein, which can for example be used for the non-therapeutic use or therapeutic use as taught herein. In addition, the spores may be combined with the at least one FAA as taught herein.

[0086] An advantage of providing health beneficial bacteria in the form of spores is that it provides for higher stability, easier mode of delivery, enhanced viability in the gut and related enhanced metabolic activity and potential gut colonisation.

[0087] Food composition

[0088] In one aspect, the present disclosure provides a food composition comprising a bacterium capable of providing a fatty acid amide (FAA) and a fatty acid amide (FAA).

[0089] In one embodiment, the food composition is obtainable or obtained by a method for providing a food composition as disclosed above.

[0090] In one embodiment, the bacterium capable of providing a fatty acid amide (FAA) is as disclosed above. In one embodiment, the bacterium capable of producing a fatty acid amide (FAA) is a Coprococcus species, preferably selected from the group consisting of Coprococcus eutactus (e.g. ATCC 27759); and Coprococcus aceti (e.g. DSM112732). Preferably, the bacterium is Coprococcus eutactus.

[0091] In one embodiment, the food composition comprises the bacterium capable of providing a fatty acid amide (FAA) as disclosed above. Preferably, the food composition comprises 10A6 to 10A11 , preferably 10A6 to 10A11 , preferably 10A8 to 10A10 colony-forming units (CFU) of the FAA-producing bacteria per gram of the food composition.

[0092] In one embodiment, the food composition comprises the bacterium capable of providing a fatty acid amide (FAA) is as disclosed above in the form of a spore. Preferably at least 15% of the bacteria are present in the form of a spore, more preferably at least 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% of all bacteria capable of providing a fatty acid amide are in the form of a spore.

[0093] In a specific embodiment, the food composition comprises Coprococcus eutactus in the form of a spore. Preferably at least 15% of the bacteria are present in the form of a spore, more preferably at least 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% of all Coprococcus eutactus bacteria are in the form of a spore.

[0094] The advantage of a food composition comprising spores is that a higher number of bacteria may be included in the food composition compared to when all of the bacteria are not in the form of spores. Further advantages are that the food composition is more robust and may be used to provide a heat treated food composition including spores of a selected bacterium with potential functional health benefits.

[0095] In one embodiment, the food composition comprises a fatty acid amide (FAA) selected from the group consisting of oleoyl dopamine; oleoyl tyramine, oleoyl aminovaleric acid, N-oleoylethanolamide, a-linolenoyl phenylethylamine, caproyl tryptamine and lauroyl tryptamine. Preferably, the fatty acid amide (FAA) is selected from the group consisting of oleoyl dopamine, oleoyl tyramine, oleoyl aminovaleric acid, and lauroyl tryptamine. More preferably, the fatty amide (FAA) is oleoyl aminovaleric acid. These particular fatty acid amides (FAAs) are more likely to induce an effect on the host. The food composition may comprise at least one, at least two, at least three, at least four, at least five, or more different fatty acid amides (FAAs) as disclosed above.

[0096] An advantage of providing a food composition comprising a bacterium capable of providing a fatty acid amide (FAA) and a fatty acid amide (FAA) is that the food composition motivates the subject to continue consumption of the food composition so that the bacteria comprised in the food composition can be active in the gut and potentially colonize the gut and subsequently produce a permanent, longer lasting positive effect on (mental) health. In this manner, an improved food product with beneficial microbes is provided.

[0097] In one embodiment, the (liquid) food composition comprises at least one fatty acid amide (FAA) as disclosed above, preferably in an amount of 0.05 pM to 2 pM of a fatty acid amide (FAA), preferably 0.1 pM to 0.8 or 1 pM, more preferably 0.2 pM to 0.6 or 0.7 pM, most preferably 0.25 to 0.5 pM.

[0098] In one embodiment, the bacterium capable of providing a fatty acid amide (FAA) provides the same fatty acid amide as the fatty acid amide (FAA) present in the food composition. For example, the bacterium may be Coprococcus eutactus capable of providing oleoyl aminovaleric acid and the fatty acid amide (FAA) present in the food composition is oleoyl aminovaleric acid.

[0099] In one embodiment, the bacterium capable of providing a fatty acid amide (FAA) provides a different fatty acid amide as the fatty acid amide (FAA) present in the composition. For example, the bacterium may be Coprococcus eutactus capable of providing oleoyl aminovaleric acid and the fatty acid amide (FAA) present in the food composition is lauroyl tryptamine.

[0100] In a preferred embodiment, the food composition according to the present disclosure comprises Coprococcus eutactus and oleoyl aminovaleric acid.

[0101] Preferably, the food composition comprises cereals, preferably oats; more preferably oats and dairy; even more preferably oats, dairy and glucose.

[0102] In one embodiment, the food composition comprises oleoyl dopamine, which is a known human GPCR-targeting FAA, and a bacterium selected from the group consisting of Marvinbryantia formatexigens, Blautia sp. Marseille-P2398, Blautia wexlerae, Lachnospiraceae str. LC2019, Clostridium sp. L2-50 and Clostridium sp. CAG:253. Each of these bacteria are capable of providing oleoyl dopamine.

[0103] The other major products are oleoyl tyramine, oleoyl aminovaleric acid, a-linolenoyl phenylethylamine, caproyl tryptamine and lauroyl tryptamine for the Ruminococcaceae bacterium and Blautia sp. An81 pathways, Coprococcus eutactus pathway, Ruminococcus albus pathway, Ruminococcus sp. CAG:488 pathway and Eubacterium rectale pathway, respectively

[0104] In one embodiment, the food composition comprises oleoyl dopamine and the bacterium Marvinbryantia formatexigens, which is capable of providing oleoyl dopamine.

[0105] In addition, or alternatively, the food composition comprises oleoyl tyramine and the bacterium Ruminococcaceae bacterium, which is capable of providing oleoyl tyramine.

[0106] In addition, or alternatively, the food composition comprises a-linolenoyl phenylethylamine and the bacterium Ruminococcus albus, which is capable of providing a- linolenoyl phenylethylamine.

[0107] In addition, or alternatively, the food composition comprises caproyl tryptamine and the bacterium Ruminococcus sp. CAG:488 capable of providing caproyl tryptamine.

[0108] In addition, or alternatively, the food composition comprises lauroyl tryptamine and Eubacterium rectale capable of providing lauroyl tryptamine.

[0109] It is also foreseen that the food composition comprises no FAAs, or less than 1 , 2, 3, 4, 5 wt.%, but comprises one or more of the bacterium according to the present disclosure, preferably in form of vegetative cells and / or spores.

[0110] In one embodiment, the food composition may comprise further ingredients, such as vitamins, minerals, flavorings, or other functional components, to enhance the nutritional profile and sensory characteristics of the final food composition Such other functional components may be further postbiotics, such as metabolites produced by fermenting microorganisms, for example butyric acid.

[0111] In certain embodiments, the composition is suitable for oral administration

[0112] In one embodiment, the food composition according to the present disclosure further comprises short chain fatty acids. Preferably, the short chain fatty acids are butyric acid. Butyric acid is known for its beneficial effects on gut health and has been shown to support the maintenance of the intestinal barrier, reduce inflammation, and promote overall digestive health. The combination of butyric acid and fatty acid amide, in particular oleoyl aminovaleric acid, is believed to have a synergistically enhanced health promoting effect.

[0113] In one embodiment, the food composition according to the present disclosure is in the form of a tablet, capsule, ampoule or powder. These forms are convenient for dosing and can be easily incorporated into daily routines. Tablets and capsules provide precise dosing and are easy to transport, while powders and ampoules can be mixed with liquids for ease of consumption.

[0114] In one embodiment, the food composition according to the present disclosure is in the form of a beverage or a food bar. A food bar refers to a food composition typically formed into a rectangular shape, designed to be eaten by hand and provide nutrition in a convenient, portable form. These forms offer a convenient and enjoyable way to consume the food composition, making it easier to integrate into daily life.

[0115] In one embodiment, the food composition is a baked food composition. The food composition obtained by baking, i.e. , exposing the food composition to a temperature of 150 °C to 250 °C, preferably 160 °C to 220 °C, more preferably 175 °C to 190 °C. Preferably the food composition is baked for at least 10 minutes, more preferably 15 to 60 minutes.

[0116] In one embodiment, the food composition is a food bar comprising bacteria capable of producing a fatty acid amide (FAA), wherein, in order of preference, at least 15%, 25%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% of the bacteria are in the form of a spore. The food bar is further preferably obtained by baking, i.e, exposing the food composition to a temperature of 150 °C to 250 °C, preferably 160 °C to 220 °C, more preferably 175 °C to 190 °C. Preferably the food composition is baked for at least 10 minutes, more preferably 15 to 60 minutes. The provision of the food composition in the form of a food bar provides the advantage of providing a more convenient manner of consuming the food composition.

[0117] Preferably, the food bar comprises oats, preferably 30-70% by weight of oats of the total food composition. Also other food products are foreseen, such as beverages, or fermented breakfast cereals.

[0118] Preferably, the food composition is not and / or does not comprise Camembert cheese.

[0119] Non-medical use

[0120] In one aspect, the present disclosure relates to a non-medical use of a fermented food composition as disclosed herein e.g. to improve mood, promote physical exercise and / or reduce stress.

[0121] In one aspect, the present disclosure relates to a non-medical use of a fermented food composition as disclosed herein e.g. to prevent poor mood and / or stress.

[0122] In one embodiment, the food composition comprises bacteria capable of producing a fatty acid amide (FAA), wherein, in order of preference, at least 15%, 25%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% of the bacteria are in the form of a spore. Preferably all of the bacteria are in the form of a spore.

[0123] In a preferred embodiment, the food composition comprises Coprococcus eutactus and at least 90%, preferably 95% of the bacteria are in the form of a spore. use

[0124] In a further aspect, the present disclosure relates to a (food) composition as disclosed herein for use as a medicament.

[0125] In one embodiment, the (food) composition as disclosed herein is for use in the treatment and / or prevention of a neurological disorder or metabolic disorder.

[0126] In one embodiment, the (food) composition as disclosed herein is for use in the treatment and / or prevention of a neurological disorder selected from the group consisting of Parkinson’s disease (PD), autism, depression, anxiety and schizophrenia.

[0127] These conditions are characterized by complex pathologies that can benefit from the supportive effects of the composition, potentially improving patient outcomes and quality of life.

[0128] In one embodiment, the food composition comprises bacteria capable of producing a fatty acid amide (FAA), wherein, in order of preference, at least 15%, 25%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% of the bacteria are in the form of a spore. Preferably all of the bacteria are in the form of a spore.

[0129] In a preferred embodiment, the food composition comprises Coprococcus eutactus and at least 90%, preferably 95% of the bacteria are in the form of a spore.

[0130] Examples

[0131] Coprococcus eutactus, and Coprococcus aceti can be isolated as described in Anaerobe Laboratory Manual of Holdeman & More (1972, Publisher: Blacksburg, Va. : The Laboratory).

[0132] Example 1 : Preparation of a fermentation matrix

[0133] Oats were autoclaved at 121 °C for 15 min and mixed with UHT whole cow’s milk under sterile conditions to provide fermentation matrices:

[0134] Fermentation matrix 1 comprised 10% oats (w / v);

[0135] Fermentation matrix 2 comprised 10% (w / v) oats (w / w) and 90% (w / w) milk;

[0136] Fermentation matrix 3 comprised 10% (w / v) oats (w / w); 90% (w / w) milk; and 0.5% (w / v) glucose;

[0137] Control fermentation matrix C1: Wilkins-Chalgren (WC) broth

[0138] Control fermentation matrix C2: Wilkins-Chalgren (WC) broth and 0.5% (w / v) glucose The liquified oats were stirred in a thermostatically controlled water bath at 80°C for 10 min to ensure homogenisation, cooled to room temperature and moved to an anaerobic environment for inoculation.

[0139] Wilkins-Chalgren (WC) broth is a specialized culture medium used for the cultivation and susceptibility testing of anaerobic microorganisms. It comprises enzymatic digests of casein and animal tissue, yeast extract, dextrose, sodium chloride, L-arginine, sodium pyruvate, hemin, and vitamin K. The pH is adjusted to 7.1 ± 0.2 at 25°C.

[0140] Example 2: Fermentation with Coprococcus eutactus

[0141] Coprococcus eutactus ATCC 27759 was grown on plates overnight and subsequently resuspended in the prepared fermentation matrix till the total liquified fermentation volume poured inside was 80% of the tube volume.

[0142] Each fermentation product was incubated at 37 °C for 24h. Five fermentation replicate experiments were performed under anaerobic conditions.

[0143] The pH was determined for each replicate directly after inoculation and after 24 hours of fermentation. There was no significant decrease in pH in the uninoculated negative control (NC) oats after 24 h incubation at 37 °C (Figure 1). In the C. ec / tactc / s-fermented oats and milk products, pH had dropped below 6.5 indicating acid production due to fermentation. C. eutactus in oats showed a pH decrease of approx. 0.5 units over 24 h of incubation at 37 °C whereas the oats products with added glucose showed a pH decrease of approx, one unit after 24 h of incubation at 37 °C.

[0144] Example 3: Enumeration of viable Coprococcus eutactus

[0145] The number of visible C. eutactus in the fermented oats-and-milk suspensions and WC broth at the start and after 24 hours of incubation at 37 °C were quantified using the dropplate method. The drop-plate method is a microbial enumeration technique that is suitable for determining colony forming units (CFUs).

[0146] To estimate the amount of live bacteria present in the developed oat product and the WC growth media, the log CFU counts per ml of media were assessed before and after 24 h of incubation at 37 °C (Figure 2). After 24 h fermentation, the C. eutactus colony count in oats and glucose increased from a log value of 5 to a log value of 10. This is a 100,000-fold increase in the number of bacteria. Increase in bacterial density in the oats and glucose medium was comparatively much higher than in the other media.

[0147] Example 4: Comparison on obtained CFU / ml, FAA level and pH

[0148] In this example, the obtained final CFU / ml, obtained final FAA level and final pH are compared upon 24 h anaerobic fermentation at 37 °C in different fermentation matrices (prepared in accordance with Example 1), i.e. Wilkins-Chalgren (WC) broth, oats (10% w / v), oats + milk (10% w / v oats) and oats+milk+glucose (10% w / v oats, 0.5% w / v glucose, remainder milk). See Table 1.

[0149] Table 1.

[0150] Example 5: Analysis of Coprococcus eutactus

[0151] To confirm the fermentation was performed using C. eutactus, the obtained bacteria after 24 hours of incubation at 37 °C were analyzed.

[0152] The bacteria were subjected to 16S rRNA sequencing, preferably using primers according to SEQ ID No. 5 and SEQ ID No. 6. It was confirmed that the bacteria were indeed C. eutactus.

[0153] Sporulation of C. eutactus is induced using ethanol or nutrient depletion.

[0154] The cell morphology and endospore structures were investigated using phase contrast microscopy (PCM) and scanning electron microscopy (SEM).

[0155] Example 6: Fermentation with Coprococcus aceti

[0156] Coprococcus aceti (strain H2_11 , DSM 107541) is cultured under anaerobic conditions on Wilkins-Chalgren (WC) agar plates and resuspended in the prepared fermentation matrices as described in Example 1. Each fermentation matrix is inoculated such that the final suspension volume fills 80% of the fermentation tubes.

[0157] Fermentation is performed at 37 °C for 24 h under anaerobic conditions. Five replicate fermentations are performed for each species. The pH is measured directly after inoculation and after 24 h of incubation. Similar to C. eutactus (Example 2), C. aceti demonstrates a reduction in pH across the tested fermentation matrices. In oats supplemented with glucose, the pH decreases by approximately one unit after 24 h, consistent with fermentation-driven acid production. In oats without glucose, the pH decrease is approximately 0.5 units, while in oats + milk the decrease is intermediate.

[0158] Example 7: Comparative analysis of CFU / ml, FAA levels, and pH for C. aceti The viable counts, FAA levels, and pH values are assessed after 24 h anaerobic fermentation at 37 °C, using the same fermentation matrices described in Example 1. Enumeration is performed using the drop-plate method, and FAA quantification is carried out using LC-MS analysis as described for C. eutactus.

[0159] Results show that C. aceti increases in CFU / ml during fermentation. In oats supplemented with glucose, bacterial density increases from approximately log 5 CFU / ml at inoculation to log 9-10 CFU / ml after 24 h. FAA production follows a similar pattern to C. eutactus, with the highest levels in the oats + milk + glucose matrix, moderate levels in oats + milk, and lower but detectable levels in oats alone. The pH profiles corresponds with those observed for C. eutactus, with final pH values between 5.7 and 6.2 depending on the matrix.

[0160] These results demonstrate that C. aceti is capable of growing in cereal-based fermentation matrices and producing FAA metabolites in a manner consistent with C. eutactus.

[0161] Table 2. Nucleic acid sequences

[0162] Table 3. Amino acid sequences

Claims

- 28 -CLAIMS1. Method for providing a food composition, wherein the method comprises the step of:- fermenting a fermentable food substrate using one or more bacterium capable of providing at least one fatty acid amide (FAA) to obtain the food composition, wherein the fermentable food substrate comprises cereals, preferably oats; more preferably oats and dairy; even more preferably oats, dairy and glucose, wherein the one or more bacterium is selected from the group consisting of Coprococcus eutactus and Coprococcus aceti, and wherein the food composition comprises at least one FAA.

2. Method for producing a food composition according to claim 1 , wherein the one or more bacterium comprises a nucleic acid comprising a nucleotide sequence with at least 70% sequence similarity to SEQ ID No. 1 ; a second nucleic acid comprising a nucleotide sequence with at least 70% sequence similarity to SEQ ID No. 2; a third nucleic acid comprising a nucleotide sequence with at least 70% sequence similarity to SEQ ID No. 3.

3. Method for producing a food composition according to any one of claims 1 to 2, wherein the bacterium is Coprococcus eutactus.

4. Method for producing a food composition according to any one of claims 1 to 3, wherein the at least one FAA is selected from the group consisting of oleoyl aminovaleric acid, and N-oleoylethanolamide (OEA), preferably oleoyl aminovaleric acid.

5. Method for producing a food composition according to any one of claims 1 to 4, wherein the fermentable food substrate comprises beta-glucan, pectin, gum acacia, inulin, or a combination of one or more of glucose, rhamnose, galalactose, xylose, arabinose, fructose, mannose, preferably beta-glucan.

6. Method for producing a food composition according to any one of claims 1 to 5, wherein the method further comprises the step of inducing the one or more bacterium to sporulate, preferably after fermenting the fermentable food substrate7. Food composition comprising- cereals, preferably oats; more preferably oats and dairy; even more preferably oats, dairy and glucose;- one or more bacterium capable of providing at least one fatty acid amide (FAA), wherein the one or more bacterium is selected from the group consisting of Coprococcus eutactus and Coprococcus aceti,- at least one fatty acid amide (FAA).

8. Food composition according to claim 7, wherein the one or more bacterium is as defined in any of claims 2 to 4.

9. Food composition according to any one of claims 7 to 8, wherein the food composition comprises at least 10A6 to 10A11 colony forming units (CFU) of said one or more bacterium per gram of the food composition.

10. Food composition according to any one of claims 7 to 9, wherein at least 15% of all of said one or more bacterium are present in the form of a spore, preferably at least 25%, more preferably at least 50%, even more preferably at least 90%.11 . Food composition according to any one of claims 7 to 10, wherein the at least one fatty acid amide (FAA) is as defined in claim 4.

12. Food composition according to any one of claims 7 to 11 , wherein the food composition comprises at least 0.1 pM to 0.8 pM of said at least one FAA, preferably 0.2 pM to 0.6 pM.

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