Extracts from fast-growing microorganisms

Abstract

The present invention relates to a method for producing a composition comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% amino acids and peptides, and a composition obtainable according to the above method, comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% amino acids and peptides. -1 The process involves obtaining a biomass by culturing at least one microbial strain characterized by a growth rate during the process. The composition of the present invention is particularly useful for the production of animal feed, pet food, food, fermentation medium or supplement, nutraceutical, pharmaceutical, diagnostic, RNA or DNA, or flavor enhancer.

Description

[Technical field]

[0001] The present invention relates to a method for producing a composition comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% amino acids and peptides, and a composition obtainable according to the above method, comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% amino acids and peptides. -1 The process involves obtaining a biomass by culturing at least one microbial strain characterized by a growth rate during the process. The composition of the present invention is particularly useful for the production of animal feed, pet food, food, fermentation medium or supplement, cosmetic, pharmaceutical, diagnostic, DNA or RNA, dietary supplement, or flavor enhancer. [Background technology]

[0002] Bacteria, for example lactic acid bacteria found in fermented dairy products such as yogurt and cheese, or in sausages, are widely used in food production, but so far they have only just begun to be used as a direct food ingredient for human or animal consumption. However, changing this would have a significant positive impact on the environment, as microbial proteins grown in fermenters could replace arable-grown proteins such as soybeans or harvested proteins such as fishmeal, reducing water, fertilizer and pesticide consumption, as well as greenhouse gas emissions. It could also lead to a reduction in land use. Next to proteins, which are essential feed and food ingredients, bacteria, especially fast-growing bacteria, contain surprisingly large amounts of nucleic acids. Nucleic acids can be converted into high concentrations of nucleotides, which can be used in food and feed production and related applications. Nucleotides have been shown to be semi-essential feed ingredients. Especially under stress conditions, the ability of organisms to produce nucleotides is limited, so an external source of this nutrient is needed. Nucleotides have a positive impact on immune system regulation and feed intake. Furthermore, in feed applications, nucleotides can replace at least a portion of antibiotics. More specifically, nucleotides can improve the effectiveness of antimicrobial solutions that are known to those skilled in the art to be less effective than antibiotics.

[0003] Furthermore, 5'-ribonucleotides, more specifically 5'-inosine monophosphate (5'-IMP) and 5'-guanine monophosphate (5'-GMP), alone or in combination with glutamic acid, have been shown to improve the taste of feeds and foods by providing a stronger umami note. It should be noted that improving the taste of feeds plays a role in feed intake, i.e. increasing the effectiveness of the feed and potentially reducing costs. Stronger umami notes occur naturally in many foods (e.g. aged cheeses, meats, soy sauce, or miso paste) or when nucleotides and glutamic acid are added to enhance the umami taste (e.g. savory products such as soups and sauces). This has been promoted for decades with yeast extracts, which have become the food additive of choice in savory products. In humans, nucleotides are mostly synthesized de novo in a cascade of energy-consuming reactions and are involved in central metabolic functions such as RNA / DNA biosynthesis, metabolic regulation, and cell signaling required during cell division or protein synthesis. Dietary supplementation with nucleotides has been shown to be beneficial for intestinal development and gastrointestinal function, and several other prebiotic effects have been reported. Of note, the use of 5'-ribonucleotides has also recently shown promising potential for reducing sugar and salt intake. 5'-ribonucleotides can also be used in the preparation of sports drinks to enhance post-exercise recovery.

[0004] One drawback of existing additives containing nucleotides is that the nucleotides are limited in concentration and therefore need to be added in large quantities for some applications, which can result in off-flavors, i.e. undesirable and / or unpleasant tastes, that may not be due to the nucleotides but to components other than the nucleotides, or impose costly processing steps to obtain concentrated nucleotide solutions.

[0005] The allowed US Pat. No. 5,399,433 relates to a composition containing at least 55% (weight / weight) (based on dry matter weight without sodium chloride) of 5'-ribonucleotides and a process for the preparation thereof.

[0006] US Pat. No. 5,399,433 discloses a method for producing aerobic single-cell protein using an autolysis process. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] European Patent No. 1587947 [Patent Document 2] International Publication No. 2016 / 161549 Summary of the Invention

[0008] The present invention relates to the production of environmentally friendly extracts containing high concentrations of proteins and nucleotides, particularly nucleotides, especially 5'-ribonucleotides, useful as additives to food, animal feed, and fermentation media. The extracts are produced from fast growing bacteria grown in fermenters using conventional growth media, or preferably aqueous media, or extracts obtained from agricultural by-streams. The present invention also relates to the specific use of bacteria with extremely high nucleic acid content (up to 40%), which offers great advantages in the production of extracts enriched in 5'-ribonucleotides, compared to conventional yeast or algae extracts, or currently available bacterial extracts. The present invention further relates to the production of extracts enriched in 5'-ribonucleotides, with growth rates of at least 0.85 h20 from fermentation processes known in the art. -1 , preferably 1.1h -1 Not only is a larger bacteria used, but also a higher dilution rate D(h) than any currently known process is used to avoid contamination with other microorganisms and further increase both process efficiency and cost-effectiveness. -1The study also focuses on the use of continuous processes in the production of microbial products. Compared to other bacteria or extracts from sources such as algae or yeast, they appear to be beneficial for applications such as taste improvement, sugar or salt reduction, antibiotic replacement in feed, or high performance fermentation additives. Of particular interest is the unique composition and growth rate of the microorganisms used, which allows to lower production costs and greatly reduce the possibility of contamination with other microorganisms.

[0009] It was a technical problem to provide improved means and methods for producing compositions with high nucleotide content, which is the subject of the present invention.

[0010] The problems described herein are solved by the embodiments described below and characterized in the claims.

[0011] The present invention is summarized in the following aspects.

[0012] In a first aspect, the present invention provides a method for producing a composition comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides, the method comprising: (a) subjecting the composition to a genomic DNA synthesis step for at least 0.85 h; -1 (b) lysing the cells present in the biomass of step (a); and (c) converting the nucleic acids present in the lysate of step (b) into nucleotides and optionally the proteins present in the lysate of step (b) into amino acids and peptides, with % being understood as % w / w of dry mass excluding NaCl.

[0013] In a particular embodiment, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides, comprises the step of: -1, preferably at least 1.2 h -1 , more preferably at least 1.4 h -1 The present invention relates to an embodiment characterized by the growth rate during the process.

[0014] In a further particular embodiment, the method for producing a composition according to the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides, relates to an embodiment, wherein the at least one microbial strain of step (a) is characterized by a nucleic acid content of at least 15%, preferably at least 20% nucleotides, more preferably at least 22%, more preferably at least 25%, even more preferably at least 28%.

[0015] Again, in a further particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which the at least one microbial strain of step (a) comprises a bacterial strain, preferably a halophilic or thermophilic bacteria, preferably the bacterial strain is characterized by a genomic G / C content of at least 40%, more preferably at least 45%, even more preferably at least 50% and most preferably at least 55%.

[0016] Again, in a more particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which the at least one microbial strain in step (a) comprises a microorganism that is a vitamin B12 autotroph.

[0017] Again, in a more particular embodiment, the method for producing the composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides, comprises the step (a) being carried out for at least 0.85 h. -1 , preferably at least 1.1 h -1 , more preferably at least 1.2 h -1 , and even more preferably at least 1.4 h -1 and the growth rate in each process of the at least one microbial strain of step (a) is limited by the dilution rate, and optionally the at least one microbial strain is cultured under non-sterile conditions.

[0018] Again, in a more particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which the at least one microbial strain in step (a) is two microbial strains, preferably two bacterial strains, more preferably at least one bacterial strain is a vitamin B12 autotroph.

[0019] Again, in a further particular aspect, the method for producing a composition according to the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which the at least one microbial strain comprises a microbial strain selected from Vibrio species, in particular Vibrio natriegens, Geobacillus species, in particular Geobacillus LC300, and Bacillus species, in particular Bacillus megaterium.

[0020] Again, in a more particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which at least one microbial strain comprises a microorganism capable of synthesizing omega-3 fatty acids.

[0021] Again, in a more particular aspect, the method for producing the composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which at least one microbial strain comprises a genetically modified microorganism.

[0022] Again, in a more particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which step (b) involves a thermal pretreatment at a temperature between 70°C and 180°C and / or for a time between 10 min and 360 min, followed by an enzymatic treatment with protease(s), preferably at a temperature between 40°C and 100°C, more preferably between 45°C and 70°C, and / or at a pH between 3.0 and 9.0, more preferably between 5.0 and 7.0, and / or for a period between 0.5 h and 72 h, more preferably between 0.5 h and 10 h.

[0023] Again, in a more particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which step (b) is carried out by mechanical homogenization, optionally with a thermal pretreatment at a temperature between 70°C and 180°C and / or for a time between 10 min and 360 min.

[0024] Again, in a more particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which step (c) involves an enzymatic treatment with nuclease(s), preferably at a temperature between 40°C and 100°C, more preferably between 45°C and 70°C, and / or at a pH between 3.0 and 9.0, more preferably between 5.0 and 7.0, and / or for a period between 0.5 hours and 72 hours, more preferably between 0.5 hours and 10 hours.

[0025] Again, in a more specific aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which step (c) further comprises an enzymatic treatment with deaminase(s), preferably at a temperature of 40°C to 100°C, more preferably at 45°C to 70°C, and / or at a pH of 3.0 to 9.0, more preferably 5.0 to 7.0, and / or for a period of 0.5 hours to 72 hours, more preferably 0.5 hours to 10 hours.

[0026] Again, in a more particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which step (c) further comprises an enzymatic treatment with protease(s), preferably at a temperature of 40°C to 100°C, more preferably at 45°C to 70°C, and / or at a pH of 3.0 to 9.0, more preferably 5.0 to 7.0, and / or for a period of 0.5 hours to 72 hours, more preferably 0.5 hours to 10 hours.

[0027] In a further aspect, the present invention relates to a composition comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% proteins and / or amino acids and peptides, obtainable according to the method for producing a composition of the present invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides.

[0028] In a particular aspect, the composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% proteins and / or amino acids and peptides relates to an embodiment in which the composition is characterized by an IMP / GMP content of at least 11%, preferably at least 13%, even more preferably at least 15% and most preferably at least 17%.

[0029] In a further particular aspect, the composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% proteins and / or amino acids and peptides relates to an embodiment in which the composition further comprises a glutamic acid content of at least 6% (wt / wt), preferably at least 8% (wt / wt), more preferably at least 10% (wt / wt), even more preferably at least 12% (wt / wt), even more preferably at least 14% (wt / wt) and most preferably at least 16% (wt / wt).

[0030] In a further aspect, the present invention relates to the use of a composition of the present invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% proteins and / or amino acids and peptides in the manufacture of an animal feed, a pet food, a fermentation medium or supplement, a food, a nutritional supplement, a pharmaceutical, a diagnostic, DNA or RNA, or a flavour enhancer.

[0031] Again, in a further aspect, the present invention relates to an animal feed, pet food, food, fermentation medium or supplement, dietary supplement or flavour enhancer comprising the composition of the present invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% proteins and / or amino acids and peptides.

[0032] In a further aspect the present invention relates to the use of a composition according to the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% proteins and / or amino acids and peptides as a replacement for monosodium glutamate or as / in a replacement for antibiotics.

[0033] Again, in a further aspect, the present invention relates to a biomass obtainable in step (a) of the process for producing the composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides.

[0034] In a particular aspect, the biomass of the invention relates to an embodiment in which the glutamic acid content is at least 5% (wt / wt), preferably at least 6% (wt / wt), more preferably at least 7% (wt / wt), even more preferably at least 8% (wt / wt), even more preferably at least 9% (wt / wt), even more preferably at least 10% (wt / wt), and most preferably at least 12%. Furthermore, the biomass of the invention preferably has a nucleic acid content of at least 15%, preferably at least 20%, more preferably at least 22% (wt / wt), more preferably at least 25% (wt / wt), and even more preferably at least 28% (wt / wt).

[0035] In a further aspect, the present invention relates to a single cell protein comprising the biomass of the present invention.

[0036] In a particular aspect, the single-cell protein of the present invention relates to embodiments, wherein the single-cell protein has further been subjected to a heat treatment.

[0037] In a further aspect the present invention relates to the use of the single-cell protein of the invention in the manufacture of animal feed, pet food or food, preferably animal feed.

[0038] In yet another aspect, the present invention relates to a cell lysate obtainable in step (b) of the process for producing the composition of the present invention, which comprises at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides.

[0039] Again, in a further aspect, the present invention relates to a composition comprising nucleotides obtainable in a process comprising a separation step of a cell lysate according to the invention, preferably by ultrafiltration.

[0040] Again, in a further aspect, the present invention relates to a composition comprising amino acids and peptides obtainable in a process comprising a separation step, preferably by ultrafiltration, of a cell lysate according to the invention.

[0041] Again, in a further aspect, the present invention relates to the use of a composition comprising nucleotides obtainable in a process comprising a separation step, preferably by ultrafiltration, of a cell lysate of the present invention, or a composition comprising amino acids and peptides obtainable by separation, preferably by ultrafiltration, of a cell lysate of the present invention, in the manufacture of an animal feed, a pet food, a food product, a fermentation medium or supplement, a nutritional supplement, a diagnostic agent, a pharmaceutical product, DNA or RNA, or a flavour enhancer.

[0042] Again, in a further aspect, the present invention relates to an animal feed, pet food, food, fermentation medium or supplement, dietary supplement, diagnostic agent, pharmaceutical agent, DNA or RNA, or flavour enhancer comprising a composition comprising nucleotides obtainable in a process comprising a separation step, preferably by ultrafiltration, of a cell lysate of the present invention, or a composition comprising amino acids and peptides obtainable by separation, preferably by ultrafiltration, of a cell lysate of the present invention.

[0043] definition The growth rate during the process is defined herein as the growth rate that can be achieved under fermentation conditions. In other words, for a microorganism to be suitable for use in the method of the present invention, it must be possible to culture the microorganism at the growth rate defined herein under fermentation conditions, particularly on a specific growth medium. The growth rate during the process defined herein refers to the growth rate of a specific microorganism, unless otherwise stated. When two or more microorganisms are co-cultured, the growth rate refers to the combined growth rate of all microorganisms in the culture, and may represent the increase in combined biomass over time.

[0044] As will be appreciated by those skilled in the art, the growth rate during the process is preferably determined by plotting the ln (natural logarithm) of biomass mass as a function of time and using linear regression to calculate the slope of the linear range, which corresponds to the exponential growth phase.

[0045] The growth rate during this process is μ(h -1 ) is also sometimes called.

[0046] As understood herein, the dilution rate D(h -1 ) is defined as the medium flow (inflow of fresh medium equal to outflow of culture) per unit time (preferably per hour) divided by the volume of culture in the reactor.

[0047] Omega-3 fatty acids as understood herein are polyunsaturated fatty acids characterized in their chemical structure by the presence of a double bond three atoms away from the terminal methyl group. In particular, the term encompasses alpha-linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid.

[0048] Nucleic acids as understood herein include deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), also understood as polydeoxyribonucleotides and / or polyribonucleotides, without particular limitation on the size of the polymer. It should be noted that nucleic acids are formed from nucleotides.

[0049] Nucleotide as understood herein refers to deoxyribonucleotide and ribonucleotide, which are monomers that form polydeoxyribonucleotide and / or polyribonucleotide, respectively, when polymerized.Note that this term also includes modified nucleotides, such as IMPs that contain inosine base.Preferably, when referring to the nucleotide content of a composition, it refers to the nucleotide, nucleoside, and nucleic acid base (pyrimidine or purine) content of the composition, and even more preferably, it refers to the nucleotide and nucleoside content of the composition. Thus, for example, a composition comprising at least 22% nucleotides, where % is understood as % w / w of dry mass excluding NaCl, is preferably understood to comprise at least 22% (understood as % w / w of dry mass excluding NaCl) of nucleotides, nucleosides, and nucleic acid bases, more preferably at least 22% (understood as % w / w of dry mass excluding NaCl) of nucleotides and nucleosides, i.e. the nucleotides, nucleosides, and nucleic acid bases together constitute at least 22% of the composition, where % is understood as % w / w of dry mass excluding NaCl, and more preferably the nucleotides and nucleosides together constitute at least 22% of the composition, where % is understood as % w / w of dry mass excluding NaCl.

[0050] A nucleoside is known to those of skill in the art as a nucleotide lacking a phosphate moiety.

[0051] Preferably, nucleobases are understood herein to include pyrimidine and purine bases, more preferably the nucleobases are adenine, cytosine, guanine, thymine and uracil.

[0052] As used herein, the term "protein" or "proteins" refers to proteins, peptides, and polypeptides, which may or may not be post-translationally modified. Post-translational modifications may be, for example, phosphorylation, methylation, glycosylation. The term "protein" is preferably used to refer to a fraction or composition comprising a protein as defined herein. Enzymatic treatment with protease(s), i.e., enzymes capable of catalyzing the hydrolysis of peptide bonds, hydrolyzes the peptide bonds in the protein, converting larger polypeptides into smaller polypeptides and / or amino acids. Thus, the term "amino acids and peptides" refers to proteins, peptides (sometimes also called polypeptides or oligopeptides), and compositions comprising amino acids, which are preferably understood herein as the products of protease treatment of "proteins". [Brief description of the drawings]

[0053] [Figure 1] A. Growth curves obtained for the growth of Vibrio natriegens on molasses and B. on glucose. CDW in this specification refers to cell dry weight. A linear regression (expressed in μ) is shown, from which the growth rate during the process can be calculated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] The means and methods of the present invention are described below, it being understood that all possible combinations of the features described herein are also contemplated.

[0055] In a first aspect, the present invention relates to a method for preparing a composition comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% amino acids and peptides. Preferably, as understood herein, reference to nucleotides may also optionally include nucleosides and nucleic acid bases (pyridine or purine), and even more preferably, reference to nucleotides may also include nucleotides and nucleosides. As understood herein, reference to "40% amino acids and peptides" may also optionally include proteins, as defined herein. As understood herein, the composition refers to the dry mass (when water is removed), further excluding sodium chloride content (NaCl). As understood herein, "%" refers to a weight / weight ratio expressed as a percentage (commonly referred to as weight / weight %). The composition referred to herein (sometimes referred to herein as the extract of the invention) comprises at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, even more preferably at least 25% nucleotides, even more preferably at least 28% nucleotides, and at least 40% amino acids and peptides, preferably at least 45% amino acids and peptides, and most preferably at least 50% amino acids and peptides. As understood herein, the composition of the invention as defined herein is obtainable according to the method of the invention.

[0056] The method of the present invention comprises: (a) providing a -1 (b) providing a biomass by culturing at least one microbial strain characterized by a growth rate during the process; (b) lysing cells present in the biomass of step (a); and (c) converting nucleic acids present in the lysate of step (b) into nucleotides and, optionally, converting proteins present in the lysate of step (b) into amino acids and peptides.

[0057] The first step (a) of the method of the present invention is carried out for at least 0.85 h. -1The objective of the present invention is to provide a biomass by culturing at least one microbial strain characterized by a growth rate during the process of 0.9 h 0.9 h 100 or more. In the method of the present invention, a fast-growing bacterial strain (with a growth rate during the process (also called growth rate) of 0.9 h 0.9 h 100 or more) is used. -1 Preferably, the at least one microbial strain in step (a) is used for at least 1.1 h. -1 , preferably at least 1.2 h -1 , more preferably at least 1.4 h -1 The present invention also provides a method for characterizing the growth rate of at least one microbial strain in step (a) during the process. -1 , at least 3h -1 , or at least 4 hours -1 Also included are embodiments characterized by a growth rate during the process of. The growth rate as understood herein is the growth rate during the process as defined herein unless otherwise specified. As will be appreciated by those of skill in the art, the growth rate during the process may vary depending on the growth medium, and may vary between minimal and rich media, as will be appreciated by those of skill in the art.

[0058] As defined herein, fast growing microorganisms are generally characterized by a high nucleic acid content compared to relatively slow growing microorganisms. The inventors have surprisingly found that the use of microorganisms with high nucleic acid content simplifies and reduces the cost of extract production, since expensive steps of nucleic acid and protein separation can be omitted. As known to those skilled in the art, a nucleotide level of at least 22% could only be achieved so far, for example when using yeast extracts, by including a further nucleic acid isolation and / or concentration step after cell lysis, as demonstrated in the prior art. Furthermore, as known to those skilled in the art, a nucleotide level of at least 15% could only be achieved so far, for example when using yeast extracts, by including a further nucleic acid isolation and / or concentration step after cell lysis, as demonstrated in the prior art. Thus, in the method of the present invention, the at least one microbial strain in step (a) is preferably characterized by a nucleic acid content of at least 15%, more preferably at least 20%, even more preferably at least 22%, even more preferably at least 25%, even more preferably at least 28%. Preferably, in the method of the invention, at least one microbial strain of step (a) is characterized by a nucleic acid content of at least 22%, preferably at least 25%, even more preferably at least 28%. In the case of yeast extracts, the nucleic acid content of yeast is lower, typically around 10%, and requires prior separation and further processing in order to reach a nucleotide content of the final extract comparable to that achievable according to the method of the invention.

[0059] Suitable examples include Vibrio natriegens strains, Geobacillus strains, and Bacillus strains. Thus, in the method of the present invention, it is preferred that the at least one microbial strain comprises a microbial strain selected from Vibrio species, in particular Vibrio natriegens, Geobacillus species, in particular Geobacillus LC300, and Bacillus species, in particular Bacillus megaterium. Preferably, the at least one microbial strain is selected from Vibrio species, in particular Vibrio natriegens. Further suitable examples of Geobacillus species may include Geobacillus uralicus, and Geobacillus stearothermophilus. Further suitable examples of Bacillus species may include Bacillus licheniformis, Bacillus coagulans, and Bacillus stearothermophilus.

[0060] Preferably, the at least one microbial strain of step (a) defined herein comprises a bacterial strain. The bacterial strain may be an extremophile. Preferably, the bacterial strain is a halophile or a thermophile. Halophiles are defined herein as organisms, particularly microorganisms, that require high concentrations of salinity, particularly NaCl, for growth and survival. Mild halophiles preferably grow at NaCl concentrations of at least 0.3M and not exceeding 0.8M. Moderate halophiles preferably grow at NaCl concentrations of at least 0.8M and not exceeding 3.4M. Extreme halophiles preferably grow at NaCl concentrations of at least 3.4M and not exceeding 5.1M. In certain embodiments, the halophiles described herein are extreme halophiles. It is further noted that certain organisms, particularly microorganisms, do not require high salt concentrations for survival but are able to tolerate such conditions. Such microorganisms may also be referred to as halotolerant organisms. The at least one microbial strain of the present invention may comprise a halotolerant organism. It is noted that in embodiments where at least one microorganism of step (a) is a halophilic or halotolerant organism, seawater can be used instead of ground water, tap water or distilled water for the preparation of the medium for the culturing step (a). In certain embodiments, said halophilic or halotolerant organisms grow faster than other microorganisms and thus may overgrow them. Therefore, in certain embodiments, seawater can be used without further sterilization, filtration and / or pasteurization. The bacterial strain as defined herein may be a thermophile. Thermophile is herein understood as an organism, in particular a microorganism, which preferably grows at high temperatures, i.e. preferably between 41°C and 122°C. It is noted that the use of halophilic and / or thermophilic microorganisms is advantageous since it significantly reduces the risk of contamination with other microorganisms.

[0061] Bacterial strains that are extremophiles, particularly halophiles or thermophiles, are typically characterized by a high genomic G / C content. Genomic G / C content is defined herein as the ratio of the amount of G and C nucleotides in the DNA of said organism to the total amount of nucleic acid bases in said organism's DNA. As understood herein, bacterial strains of the invention, preferably halophiles or thermophiles, are characterized by a genomic G / C content of at least 40%, more preferably at least 45%, even more preferably at least 50%, and most preferably at least 55%. As pointed out above, G is the only naturally occurring nucleic acid base that contributes significantly to the savory taste enhancement properties, especially the strong umami taste. Thus, an increase in G / C content can be directly translated into an increase in the abundance of 5'-guanine monophosphate (5'-GMP) in the extracts of the invention, resulting in a stronger umami taste in the extracts of the invention. Furthermore, less enzymes, especially deaminases, are required to obtain the stronger umami taste of the extracts of the invention, which also leads to cost savings.

[0062] The at least one microbial strain of the present invention may comprise a microorganism that is a vitamin B12 autotroph. The presence of vitamin B12 in food products is necessary and desirable, especially for vegan diets and / or products suitable for vegan diets. The microorganism that is a vitamin B12 autotroph is preferably Bacillus megaterium. It is noted that vitamin B12 autotrophs are considered interesting as natural sources of vitamin B12 in the organic agriculture industry.

[0063] It should be noted herein that within the scope of the present invention, the at least one microbial strain is not necessarily limited to a single microbial strain. Within the scope of the method of the present invention, the at least one microbial strain may be two, three, four or more bacterial strains. In certain preferred embodiments, the at least one microbial strain in step (a) is two microbial strains, preferably two bacterial strains. In certain preferred embodiments where the at least one microbial strain is two bacterial strains, the at least one bacterial strain is a vitamin B12 autotroph, preferably Bacillus megaterium.

[0064] In certain embodiments of the present invention, at least one microbial strain preferably comprises a genetically modified microorganism.The genetically modified organism may include, but is not limited to, genetic modifications to improve growth rate, biomass yield, and / or induce the production of certain chemicals.In certain embodiments of the present invention, at least one microbial strain comprises a microorganism capable of synthesizing omega-3 fatty acids.

[0065] According to the invention, the biomass of the invention is at least 0.85 h -1 The at least one microbial strain can be obtained by culturing at least one microbial strain characterized by a growth rate during the process. As understood herein, the at least one microbial strain is cultivated on a substrate. The skilled person can select, adjust and / or apply a specific medium(s) for the at least one microbial strain used in the present invention. The substrate used can be any commercially available medium containing a carbon source and a nitrogen source, as well as trace elements and vitamins. The substrate is preferably one or several side streams from the industry that are extracted by thermal and / or enzymatic methods or that are used directly in the fermentation and contain either proteins, C5 sugars, C6 sugars, or a combination thereof. One or more side streams can then be supplemented with other compounds to obtain a suitable growth medium (e.g. additional sugars as a carbon source or a nitrogen source such as yeast extract, as well as vitamins and trace elements). As known to the skilled person familiar with industrial fermentation processes, it is preferable to sterilize the medium before inoculating the fermenter.

[0066] As known to those skilled in the art, fermentation can be carried out as a batch process, a fed-batch process or a (semi-)continuous process.

[0067] Batch processes are characterized by the absence of any inflow of material into the fermentation vessel. In a batch process, all nutrients are provided at the beginning of the culture and no more are added in the subsequent bioprocess. No additional nutrients are added throughout the bioprocess, except for gas, acid, and base. In certain embodiments, an antifoam agent may be added. The bioprocess then continues until one of the nutrients required for growth becomes limiting. This strategy is suitable for rapid experiments such as strain characterization or nutrient medium optimization. The drawback of this convenient method is the limited yield of biomass and products. Usually, the microorganisms are not in the exponential growth phase for a long time, since the carbon source and / or oxygen transfer are the limiting factors. Once the bioprocess run in batch mode is finished, only the biomass or medium is harvested and appropriately processed to obtain the desired product. From the bioreactor's point of view, the process is repeatedly interrupted by cleaning and, if necessary, sterilization steps, and biomass is only produced in stages.

[0068] In fed-batch processes, substrates, nutrients, and other substances may be added (preferably in the form of concentrated solutions) to the fermentation vessel to extend the possible cultivation time or to increase the yield, among others. The advantage of feeding during cultivation is that a higher product amount can be achieved overall. Under certain growth conditions, the microorganisms and / or cells always double, and therefore follow an exponential growth curve. Therefore, in certain embodiments, the feed rate can also be increased exponentially. Generally, the substrate is delivered from a feed bottle (or a feed tank) to the cultivation vessel, for example via silicone tubing (or sterilizable tubing). The user can set the feed manually at any time (linear, exponential, pulsed) or add nutrients when certain conditions are met (such as when a certain biomass concentration is reached or when nutrients are depleted). Fed-batch processes offer a wide range of control strategies and are suitable for highly specialized applications. However, they can lead to long processing times and accumulation of toxic by-products, which can lead to inhibition. High cell densities can also be a constraint due to limited oxygen transfer from the gas phase.

[0069] In the method of the invention, the submerged fermentation is preferably carried out as a continuous process. After a batch growth phase, an equilibrium is established for certain components (also called steady state). Under these conditions, fresh medium is added (chemostat) as much as is withdrawn. These bioprocesses are called continuous cultures and are particularly suitable when excess nutrients are inhibited, for example due to acid or ethanol accumulation or excessive heating. Other advantages of this method include less inhibition of the product and improved space-time yield. Since the cells are harvested when the medium is withdrawn, the inflow and outflow rates must be less than the doubling time of the microorganism. Alternatively, the cells can be retained in various ways (e.g. spin filters), which is called perfusion. In a continuous process, the space-time yield of the bioreactor can be further improved compared to a fed-batch process. However, a long culture period also increases the risk of contamination and long-term changes in the culture. The three most common types of continuous cultures are chemostat (the rate of addition of a single growth-limiting substrate controls cell growth), turbidostat (an indirect measurement of cell number (turbidity or optical density) requires additional sensors but is driven by real-time feedback to control the addition and removal of liquid), and perfusion (this type of continuous bioprocessing mode is based on either retaining cells in the bioreactor or recycling cells to the bioreactor where fresh medium is provided and cell-free supernatant is removed at the same rate).

[0070] Therefore, it is preferred that step (a) is carried out as a continuous process. Preferably, the process lasts for at least 0.85 h. -1 , preferably at least 1.1 h -1 , more preferably at least 1.2 h -1 , and even more preferably at least 1.4 h -1The dilution rate is 0.05-0.15, and the growth rate of each of the at least one microbial strains in step (a) during the process is limited by the dilution rate. The dilution rate is so fast that it exceeds the growth rate of most potential contaminants. The dilution rate is so fast that most microorganisms that may be potential contaminants are automatically flushed out of the fermenter before they can grow in the fermenter. As a result, the cultivation may be carried out under non-sterile conditions. Thus, in certain embodiments of the present invention, step (a) is carried out continuously. Preferably, the process is carried out for at least 0.85 h. -1 , preferably at least 1.1 h -1 , more preferably at least 1.2 h -1 , and even more preferably at least 1.4 h -1 The dilution rate is 0.01 to 0.01, and the growth rate during each process of the at least one microbial strain in step (a) is limited by the dilution rate, and optionally the at least one microbial strain is cultured under non-sterile conditions. Non-sterile conditions are understood herein as conditions in which the fermenter is not sterilized, such as by autoclaving, and / or the medium is not sterilized by autoclaving, filtration, or any other method known to those skilled in the art. As understood herein, in certain embodiments, the medium may be sterilized or pasteurized, but the fermenter may be used without sterilization. It is further noted that under these conditions, other microorganisms (other than the at least one microbial strain of the present invention) can grow, but the at least one microbial strain of the present invention will outgrow them (or outgrow them).

[0071] In one embodiment, step (a) is carried out as a continuous fermentation of at least one microbial strain for at least 0.85 h. -1 , preferably at least 1.1 h -1 , more preferably at least 1.2 h -1 , and even more preferably at least 1.4 h -1 In one embodiment, step (a) is performed as a continuous fermentation of the at least one microbial strain at a dilution rate of at least 0.85 h.-1 , preferably at least 1.1 h -1 , more preferably at least 1.2 h -1 , and even more preferably at least 1.4 h -1 and at least one microbial strain comprises a vitamin B12 autotroph, preferably Bacillus megaterium.

[0072] In one embodiment, step (a) involves recycling cells of at least one microbial strain.

[0073] In certain embodiments, step (a) of the present invention may involve the co-cultivation of two or more microbial strains, where at least one strain is cultured for at least 0.85 h. -1 Such an embodiment does not demonstrate an in-process growth rate of at least one microbial strain of at least 0.85 h. -1 , preferably at least 1.1 h -1 , more preferably at least 1.2 h -1 , and even more preferably at least 1.4 h -1 When two or more microorganisms are co-cultured, the growth rate refers to the combined growth rate of all the microorganisms in the culture, which may represent the increase in combined biomass over time.

[0074] Once the cultivation is complete, the biomass is harvested according to methods known to those skilled in the art. The harvesting of the biomass may include separation and / or washing. State of the art separation techniques employ tangential flow filtration or industrial (nozzle or centrifugal) separators. The biomass obtainable as described herein has a dry mass of 10% to 25% (defined as a weight to weight ratio expressed as a percentage, % (wt / wt)).

[0075] The biomass obtainable herein is used in step (b) of the method of the invention to produce a composition comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides. However, said biomass can also be used in other processes and applications as conceivable by the skilled artisan. It is therefore noted that in another aspect, the invention relates to the biomass obtainable herein.

[0076] Step (b) of the method of the invention for producing a composition comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides, comprises lysing the cells present in the biomass of step (a). Any industrially applicable method known to the skilled artisan can be used herein. The production of an extract within the scope of the invention can be achieved, for example, by mechanical or chemical disruption followed by various enzymatic treatments and by inactivation of the biomass prior to enzymatic treatment with various enzymes.

[0077] In certain embodiments, mechanical disruption methods such as bead mill or PEF are used, especially when at least one microbial strain is a gram-negative bacterium. Chemical methods of cell disintegration can also be used within the scope of the present invention. It should be noted that the methods of lysing cells discussed herein can be adapted based on the microorganism. For example, when using one or more gram-negative bacteria, lysis is easier than in the case of gram-positive bacteria, such as Geobacillus strains or Bacillus megaterium. Furthermore, compared to yeast extracts or plant cells, bacteria have thin cell walls that are more easily broken during the lysis process (particularly true for gram-negative bacteria such as Vibrio natriegens). It should be noted that lysis of such cells is more efficient than lysis of yeast or plant cells.

[0078] Chemical methods of cell disruption include, but are not limited to, treatment with salt(s), basic reagent(s), detergent(s), and / or detergent(s). It should be noted that chemical methods, especially those using basic reagent(s), also called alkaline reagent(s), can lead to partial degradation of nucleic acids, e.g., degradation of RNA, formation of 2'-ribonucleotides and 3'-ribonucleotides.

[0079] Optionally, a thermal pretreatment of the biomass can be performed prior to dissolution or homogenization. The thermal pretreatment is typically performed at a temperature between 70°C and 180°C and / or for a time between 10 minutes and 360 minutes. A person skilled in the art can adjust the conditions of the thermal pretreatment to suit the biomass in question and the experimental environment. Whether or not a thermal pretreatment is necessary depends on the further processing planned. When the biomass is dissolved by mechanical homogenization as described herein, it is preferable to perform a thermal pretreatment. Without wishing to be bound by theory, it is noted that the purpose of the thermal pretreatment is to inactivate the enzymes of at least one microbial strain. When the biomass is dissolved by mechanical homogenization, it is preferable to keep the thermal pretreatment step short, preferably less than 30 minutes, more preferably less than 15 minutes. As understood herein, the duration of the thermal pretreatment is longer.

[0080] Thus, in a particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides relates to an embodiment in which step (b) is carried out by mechanical homogenization, optionally with a thermal pretreatment at a temperature between 70°C and 180°C and / or for a period between 10 minutes and 360 minutes.

[0081] Alternatively, cell lysis can be achieved by autolysis, a process in which a microorganism destroys itself when the temperature becomes higher than its optimum, involving the production by the microorganism of specific enzymes that destroy proteins or other components that are denatured at high temperatures, for example.

[0082] In the route involving autolysis, the biomass undergoes a process of autolysis induced by heat treatment at temperatures between 45°C and 200°C for 0.1 to 48 hours. Anyone familiar with microbial autolysis technology will know that the temperature and incubation must be selected according to the nature of the microorganisms mentioned above, for example, thermophilic and mesophilic bacteria have different autolysis temperatures, and gram-positive and gram-negative bacteria require different incubation times due to the different cell wall structures. Similarly, treatment of the same biomass for different times or at different temperatures will produce extracts with different compositions.

[0083] Preferably, in the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides, the cell lysis in step (b) is not carried out by autolysis.

[0084] For pathways involving mechanical or chemical disruption followed by various enzymatic treatments, in a first step the biomass is subjected to mechanical or chemical disruption to break down the cell walls, and then the resulting cellular components are directly treated with an enzyme cocktail comprising proteolytic enzymes, nucleases, AMP deaminases, or combinations thereof.

[0085] As understood herein, the composition of the present invention may, in certain embodiments, contain cell walls.However, the present invention also encompasses compositions from which cell walls have been removed according to methods known to those skilled in the art.Preferably, the removal of cell walls is performed together with or after step (b) of the method of the present invention.

[0086] As known to the skilled artisan, steps (b) and (c) may involve autolysis of the cells of at least one microbial strain contained in the biomass of step (a), preferably at a temperature between 40°C and 200°C and / or at a pH between 3.0 and 9.0, and / or for a period between 10 minutes and 72 hours. In certain embodiments, the method for producing the composition of the invention, in which steps (b) and (c) involve autolysis of the cells of at least one microbial strain contained in the biomass of step (a), may result in a composition comprising at least 1.5% nucleotides, preferably at least 2.5% nucleotides, more preferably at least 3.5% nucleotides, even more preferably at least 5% nucleotides. The final nucleotide content depends, inter alia, on the nucleotide content of the biomass of step (a). It is understood that when steps (b) and (c) involve autolysis of the cells of at least one microbial strain contained in the biomass of step (a), a thermal pretreatment step is not required. Therefore, in these embodiments, it is preferred not to carry out the thermal pretreatment step disclosed herein.

[0087] In a particular embodiment of the present invention, in the method for producing a composition of the present invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides, step (b) comprises a thermal pretreatment followed by an enzymatic treatment with protease(s). As understood herein, the thermal pretreatment step is preferably carried out at a temperature between 70°C and 180°C and / or for a time between 10 minutes and 360 minutes. The enzymatic treatment with protease(s) is preferably carried out at a temperature between 40°C and 100°C, more preferably at a temperature between 45°C and 70°C. The enzymatic treatment with protease(s) is further preferably carried out at a pH between 3.0 and 9.0, more preferably between 5.0 and 7.0. The enzymatic treatment with protease(s) is preferably carried out for a time between 0.5 hours and 72 hours, more preferably between 0.5 hours and 10 hours. It is noted that, as described herein, the enzymatic treatment is preferably carried out after the thermal pretreatment. Any protease enzyme known to a person skilled in the art can be used for the enzymatic treatment with the protease(s) described herein. A person skilled in the art can select the appropriate protease or protease cocktail, also called proteases, and can also determine the optimal dosage of the protease(s). It is preferred to use 0.01% to 5% (w / w) of protease based on the total weight of biomass.

[0088] Those skilled in the art know that some RNA in cells forms complexes with proteins, especially RNA polymerase and sigma factors. Therefore, not all RNA is readily accessible to nucleases, which may prevent complete conversion of RNA to nucleotides and nucleosides. Appropriate treatment with protease prior to nuclease enzyme treatment may degrade RNA-protein complexes and improve nucleotide yield.

[0089] Furthermore, it should be noted that during the thermal pretreatment and subsequent enzymatic treatment with protease(s), the cell walls remain in the reaction mixture and preferably need to be separated. Separation can be achieved by mechanical and / or physical separation. Without wishing to be bound by theory, it is understood herein that the thermal pretreatment followed by enzymatic treatment with protease(s) leads to cell lysis.

[0090] The inventors have surprisingly found that the nucleic acid content in the microbial strain(s) used in the present invention, particularly bacteria, is so high that it is not necessary to separate the nucleic acids from other cellular components to produce an extract rich in nucleotides. In other words, the inventors have demonstrated that the use of microbial strain(s) with high nucleic acid content allows for the production of extracts with a nucleotide content that would otherwise only be obtainable by processes involving a step(s) of nucleic acid separation and / or concentration.

[0091] In certain embodiments of the present invention, an optional step of the method of the present invention can involve the separation of nucleic acids, and optionally proteins, from other components of the obtained extract / lysate. Preferably, said separation can be carried out using state-of-the-art ultrafiltration techniques with a cut-off between 10 kD and 50 kD. Alternatively, magnetic beads can be used to produce extracts with higher nucleotide concentrations or purer nucleotides of interest.

[0092] In both embodiments, the nucleic acid is recovered in the retentate, but in the latter case, the nucleic acid specifically binds to the beads and is subsequently eluted, leading to obtaining a nucleic acid fraction of high purity. In the case of ultrafiltration, it may be expected to perform additional purification steps to increase the purity of the recovered nucleic acid, as known to those skilled in the art. In certain alternative embodiments, an extract with a higher nucleotide concentration or a substantially pure nucleotide of interest can be obtained by chromatography, in particular ion exchange chromatography. In certain embodiments, an extract with a higher nucleotide concentration can be obtained by removing impurities during activated carbon treatment. In certain alternative embodiments, an extract with a higher nucleotide concentration or a substantially pure nucleotide of interest can be obtained by crystallization. In certain embodiments, an extract with a higher nucleotide concentration or a substantially pure nucleotide of interest can be obtained by a means involving a combination of the means disclosed above.

[0093] Again, in a further particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides, relates to an embodiment in which step (c) involves an enzymatic treatment with nuclease(s). The enzymatic treatment with nuclease is preferably carried out at a temperature between 40°C and 100°C, more preferably at a temperature between 45°C and 70°C. The enzymatic treatment with nuclease is preferably carried out at a pH between 3.0 and 9.0, more preferably between 5.0 and 7.0. The enzymatic treatment with nuclease is preferably carried out for a period between 0.5 hours and 72 hours, more preferably between 0.5 hours and 10 hours. As described herein, any nuclease enzyme known to the skilled artisan can be used for the enzymatic treatment with nuclease(s). The skilled artisan can also select the appropriate nuclease or nuclease cocktail, also called nucleases, and determine the optimal amount of nuclease(s) to be added. The nuclease is preferably used in an amount of 0.01 to 5% (weight / weight) based on the total weight of the lysate.

[0094] Again, in a further particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides, relates to an embodiment in which step (c) further comprises an enzymatic treatment with deaminase(s). The enzymatic treatment with deaminase(s) is preferably carried out at a temperature between 40°C and 100°C, more preferably at a temperature between 45°C and 70°C. The enzymatic treatment with deaminase(s) is preferably carried out at a pH between 3.0 and 9.0, more preferably at a pH between 5.0 and 7.0. The enzymatic treatment with deaminase(s) is preferably carried out for a period between 0.5 hours and 72 hours, more preferably between 0.5 hours and 10 hours. As described herein, any deaminase enzyme known to the skilled artisan can be used for the enzymatic treatment with deaminase(s). The skilled artisan can select suitable deaminases or deaminase cocktails, also called deaminases. The deaminase is preferably used in an amount of 0.01 to 5% (weight / weight) based on the total weight of the lysate.

[0095] According to the invention, the steps of enzymatic treatment with nuclease(s) and deaminase(s) can be performed separately, sequentially or together. In the case of sequential applications, an additional step of thermally inactivating the first enzyme used before adding the second enzyme can be included, as known to the skilled person. Preferably, the steps of enzymatic treatment with nuclease(s) and deaminase(s) are performed together. Such treatment can be referred to as treatment with nuclease(s) and deaminase(s). The selection of the specific nuclease(s) and deaminase(s) is performed by the skilled person such that under the treatment conditions the enzymes exhibit a specific activity and preferably cannot exhibit non-specific activity. As discussed herein, the treatment with nuclease(s) and deaminase(s) is preferably performed at a temperature between 40°C and 100°C, more preferably between 45°C and 70°C. The treatment with nuclease(s) and deaminase(s) is preferably carried out at a pH of 3.0 to 9.0, more preferably at a pH of 5.0 to 7.0. Furthermore, the treatment with nuclease(s) and deaminase(s) and the enzymatic treatment with deaminase(s) are preferably carried out for 0.5 to 72 hours, more preferably for 0.5 to 10 hours. For example, an enzyme that can withstand high salt concentrations (e.g., NaCl) of more than 3% (wt / wt), more than 5% (wt / wt), more than 7% (wt / wt), or more than 9% (wt / wt) is particularly preferred.

[0096] Plant proteins from various sources such as pea, soybean, or wheat have been introduced as replacements for animal proteins, both for human consumption and animal feed, but they have several drawbacks. First, most plant materials do not contain complete proteins (understood according to the FAO definition, www.fao.org, https: / / en.wikipedia.org / wiki / Complete_protein, accessed 11 March 2021) and often contain antinutritional compounds (such as saponins). Incomplete proteins and antinutrients reduce the effectiveness of the feed or create the need to supplement animal proteins such as fish or whey in animal feed. In human food, they can cause amino acid deficiencies in vegan-based diets. Second, the protein concentration of plants is relatively low (5%-30%) and the proteins are contained in rigid plant cell structures. Therefore, energy-demanding equipment such as jet or air classifiers is required to separate the protein fractions and concentrate them to 80% of the live protein content. The present invention overcomes these challenges by cultivating fast growing microorganisms, particularly bacteria, that contain high concentrations of intact protein (40% or more) and have thinner cell walls.

[0097] Again, in a more particular aspect, the method for producing a composition of the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides, relates to an embodiment in which step (c) further comprises an enzymatic treatment with protease(s). The enzymatic treatment with protease(s) is preferably carried out at a temperature between 40°C and 100°C, more preferably at a temperature between 45°C and 70°C. The enzymatic treatment with protease(s) is preferably carried out at a pH between 3.0 and 9.0, more preferably at a pH between 5.0 and 7.0. Furthermore, the enzymatic treatment with protease(s) is preferably carried out for a period between 0.5 hours and 72 hours, more preferably between 0.5 hours and 10 hours. As described herein, any protease enzyme known to the skilled artisan can be used for the enzymatic treatment with protease(s). The skilled artisan can select suitable proteases or protease cocktails, also called proteases. The protease(s) as defined herein preferably comprise alcalase and / or papain. Furthermore, the skilled artisan can determine the optimal dosage of the protease(s). The protease is preferably used at 0.01% to 5% (w / w) based on the total weight of the lysate.

[0098] The product of step (c) may further undergo additional processing, as known to those skilled in the art. The composition may further be formulated with other compositions and / or supplements, proteins, nucleic acids, vitamins. Odor neutralization may need to be performed according to methods known to those skilled in the art. A suitable technique for this is filtration of the composition or a solution of the composition through an activated carbon filter. The composition may further be sterilized, for example by applying a pulsed electric field, as known to those skilled in the art. In one embodiment, the composition may further be pasteurized, for example by high pressure pasteurization. The composition of the present invention may further undergo drying and moisture removal, for example by spray drying, band drying, or drum drying.

[0099] In a further embodiment, the present invention also refers to a composition comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% proteins and / or amino acids and peptides, obtainable according to the method for producing a composition of the present invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% amino acids and peptides. Preferably, said composition is characterized by an IMP / GMP content of at least 11%, preferably at least 13%, even more preferably at least 15%, most preferably at least 17%. IMP and GMP are the main nucleotides contributing to the taste properties and / or taste enhancing properties of the composition of the present invention.

[0100] In certain embodiments, the composition further comprises additional compounds that may contribute to the taste characteristics and / or taste enhancing properties of the composition of the present invention. One such compound known to those skilled in the art is glutamic acid. As used herein, glutamic acid is understood as any form of glutamic acid or its salts that is encompassed by this definition. As used herein, glutamine is also understood to be included in the term "glutamic acid". Thus, as used herein, the term glutamic acid refers to glutamic acid or its salts, or glutamine or its salts. Thus, in certain embodiments, the composition further comprises a glutamic acid content of at least 6% (w / w), preferably at least 8% (w / w), more preferably at least 10% (w / w), even more preferably at least 12% (w / w), even more preferably at least 14% (w / w), and most preferably at least 16% (w / w). Without wishing to be bound by theory, it is noted that the main source of glutamic acid is the amino acids and peptides present in the composition of the present invention.

[0101] The composition of the present invention, comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% proteins and / or amino acids and peptides, is useful for the manufacture of further products, including food. A food or feed product is defined herein as any product suitable for oral intake, preferably a food, feed, beverage, or food or feed supplement. Thus, the food or feed product preferably has a taste acceptable to the intended animal species. Thus, the composition of the present invention is useful for the manufacture of animal feed, pet food, fermentation medium or supplement, food, dietary supplement, pharmaceutical, diagnostic, DNA or RNA, or flavor enhancer.

[0102] The composition of the present invention, comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% protein and / or amino acids and peptides, is useful for the manufacture of animal feed or pet food. Animal feed is defined herein as food intended for the nutrition of preferably farmed animals, especially livestock. It can be produced, for example, in solid form, for example in the form of pellets, or in any other form suitable for feeding animals. Pet food, as defined herein, is intended for small farmed animals, such as dogs, cats, mice, guinea pigs, domestic birds, ornamental fish, etc. However, this list is not intended to be limiting in any way.

[0103] The composition of the present invention, comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% protein and / or amino acids and peptides, is useful for the manufacture of fermentation media or supplements. Fermentation media is understood herein as a substrate or nutrient source on which microbial strain(s) can grow and can be cultured in a controlled manner, in particular for laboratory or biotechnological purposes. Fermentation supplements are understood herein as products that are added to fermentation media, but do not necessarily need to be able to support the growth of microbial strains on their own.

[0104] The composition of the present invention, comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% protein and / or amino acids and peptides, is useful for the manufacture of dietary supplements.As understood herein, dietary supplements, also called functional foods, are defined as any food that goes beyond mere nutrition and has at least one specific target action to improve the health and / or well-being of the host and / or prevent a pathological condition in the host.

[0105] Again, in a further aspect, the present invention relates to an animal feed, pet food, food, fermentation medium or supplement, dietary supplement or flavour enhancer comprising a composition of the present invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% protein and / or amino acids and peptides. In other words, the present invention relates to an animal feed, pet food, food, fermentation medium or supplement, dietary supplement or flavour enhancer produced using a composition of the present invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides, and at least 40% protein and / or amino acids and peptides.

[0106] In a further aspect the present invention relates to the use of a composition comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% proteins and / or amino acids and peptides as a replacement for monosodium glutamate or as / in a replacement for antibiotics.

[0107] In a further aspect the present invention relates to the use of a composition according to the invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% proteins and / or amino acids and peptides for the manufacture of a pharmaceutical, a diagnostic, DNA or RNA.

[0108] In yet another aspect, the present invention also relates to a biomass obtainable in step (a) of the method for producing a composition of the present invention comprising at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides. As understood herein, a biomass of the present invention comprises a biomass having a molecular weight of at least 0.85 h. -1 The method can be obtained by culturing at least one microbial strain characterized by a growth rate during the process of (a). Preferably, the at least one microbial strain in step (a) is cultured for at least 1.1 h. -1 , preferably at least 1.2 h -1 , more preferably at least 1.4 h -1 The growth rate as understood herein is the growth rate during the process as defined herein, unless otherwise specified. As understood herein, in certain embodiments, the culturing is carried out as a process. Preferably, the process is characterized by a growth rate during the process of at least 0.85 h. -1 , preferably at least 1.1 h -1 , more preferably at least 1.2 h -1 , and even more preferably at least 1.4 h -1and the growth rate of each of the at least one microbial strains during the process is limited by the dilution rate. Suitable microbial strains include, but are not limited to, Vibrio natriegens, Geobacillus and Bacillus strains. Thus, the biomass of the present invention preferably comprises a microbial strain selected from Vibrio natriegens, Geobacillus and Bacillus strains. In a further preferred embodiment, the glutamic acid content is at least 5% (w / w), preferably at least 6% (w / w), more preferably at least 7% (w / w), even more preferably at least 8% (w / w), even more preferably at least 9% (w / w), even more preferably at least 10% (w / w), and most preferably at least 12% (w / w).

[0109] In a further aspect, the present invention relates to a single-cell protein comprising the biomass of the present invention. Biomass is as defined herein and above. As understood herein, single-cell protein refers to an edible microorganism, preferably a single-cell microorganism. The single-cell protein of the present invention is useful for the manufacture of animal feed, pet food or food, preferably animal feed. The single-cell protein as defined herein can be further subjected to heat treatment as described herein. Without wishing to be bound by theory, it is noted that the heat pretreatment can reduce the content of nucleic acids to a level acceptable for food manufacture or consumption as food. In certain embodiments, heat treatment can be understood as inducing autolysis in the cells forming the single-cell protein, as described herein.

[0110] In yet another aspect, the present invention relates to a cell lysate obtainable in step (b) of the process for producing the composition of the present invention, which comprises at least 15% nucleotides, preferably at least 18% nucleotides, more preferably at least 22% nucleotides and at least 40% amino acids and peptides.

[0111] In certain embodiments, the present invention relates to a cell lysate obtainable by the method described herein, wherein steps (b) and (c) involve autolysis of cells of at least one microbial strain comprised in the biomass of step (a).

[0112] Again, in a further aspect, the present invention relates to a composition comprising nucleotides obtainable by separation of a cell lysate according to the invention, preferably by ultrafiltration. Preferably, the composition comprising nucleotides comprises at least 22% nucleotides, preferably at least 25% nucleotides, more preferably at least 30% nucleotides. Preferably, the composition comprising nucleotides as defined herein is characterized by an IMP / GMP content of at least 11%, preferably at least 13%, even more preferably at least 15%, most preferably at least 17%.

[0113] Again, in a further aspect, the present invention relates to a composition comprising amino acids and peptides obtainable by separation of the cell lysate of the present invention, preferably by ultrafiltration or using magnetic beads, more preferably by ultrafiltration. In certain aspects, the separation step, as understood herein, may be understood as a concentration step.

[0114] Again, in a further aspect, the present invention relates to the use of a composition comprising nucleotides obtainable in a process comprising a separation step, preferably by ultrafiltration, of a cell lysate of the present invention, or a composition comprising amino acids and peptides obtainable in a process comprising a separation step, preferably by ultrafiltration, of a cell lysate of the present invention, in the manufacture of an animal feed, a pet food, a food product, a fermentation medium or supplement, a nutritional supplement, a pharmaceutical product, a diagnostic agent, a DNA or RNA, or a flavor enhancer. As understood herein, a separation step relates to the separation of the nucleic acids and proteins respectively contained in the lysate product of step (b) of the method of the present invention, typically followed by a step (c) of converting the nucleic acids present in the lysate of step (b) into nucleotides and optionally converting the proteins present in the lysate of (b) into amino acids and peptides. As understood herein, a separation step may also be understood as a concentration step. Preferably, a composition comprising nucleotides as defined herein is characterized by an IMP / GMP content of at least 11%, preferably at least 13%, more preferably at least 15% and most preferably at least 17%. IMP and GMP are the main nucleotides contributing to the taste properties and / or taste enhancing properties of the composition of the present invention. It is further noted that in certain embodiments, compositions comprising the nucleotides described herein and compositions comprising the amino acids and peptides described herein may be used together in the manufacture of animal feed, pet food, food, fermentation media or supplements, dietary supplements, pharmaceuticals, diagnostics, DNA or RNA, or flavor enhancers.

[0115] Again, in a further aspect, the present invention relates to an animal feed, a pet food, a food product, a fermentation medium or a supplement, a dietary supplement, or a flavor enhancer comprising a composition comprising nucleotides obtainable in a process comprising a separation step of a cell lysate of the present invention, preferably by ultrafiltration or by magnetic beads, more preferably by ultrafiltration, or a composition comprising amino acids and peptides obtainable in a process comprising a separation step of a cell lysate of the present invention, preferably by ultrafiltration or by magnetic beads, more preferably by ultrafiltration. The process comprising a separation step is as described above. As understood herein, the separation step relates to the separation of nucleic acids and proteins respectively contained in the lysate product of step (b) of the method of the present invention, typically followed by a step (c) of converting the nucleic acids present in the lysate of step (b) into nucleotides and optionally converting the proteins present in the lysate of (b) into amino acids and peptides.

[0116] Further aspects and / or embodiments of the present invention are disclosed in the following numbered paragraphs:

[0117] 1. A method for producing a composition comprising at least 22% nucleotides and at least 40% amino acids and peptides, comprising: (a) At least 0.85 hours -1 Providing a biomass by culturing at least one microbial strain characterized by a growth rate during the process; (b) lysing the cells present in the biomass of step (a); (c) converting the nucleic acids present in the lysate of step (b) into nucleotides and, optionally, converting the proteins present in the lysate of (b) into amino acids and peptides; where % is understood to be % w / w of dry mass excluding NaCl.

[0118] 2. At least one microbial strain of step (a) is incubated for at least 1.1 h. -1 , preferably at least 1.2 h -1 , more preferably at least 1.4 h -1The method according to item 1, characterized by a growth rate during the process.

[0119] 3. The method according to item 1 or 2, wherein at least one microbial strain of step (a) is characterized by a nucleic acid content of at least 22%, preferably at least 25%, even more preferably at least 28%.

[0120] 4. The method according to any one of items 1 to 3, wherein the at least one microbial strain in step (a) comprises a bacterial strain, preferably a halophile or a thermophile, preferably the bacterial strain is characterized by a genomic G / C content of at least 40%, more preferably at least 45%, even more preferably at least 50%, most preferably at least 55%.

[0121] 5. The method according to any one of items 1 to 4, wherein at least one microbial strain in step (a) comprises a microorganism that is a vitamin B12 autotroph.

[0122] 6. Step (a) is carried out for at least 0.85 h. -1 , preferably at least 1.1 h -1 , more preferably at least 1.2 h -1 , and even more preferably at least 1.4 h -1 and the growth rate in each process of the at least one microbial strain of step (a) is limited by the dilution rate, and optionally the at least one microbial strain is cultured under non-sterile conditions.

[0123] 7. The method according to any one of items 1 to 6, wherein the at least one microbial strain in step (a) is two microbial strains, preferably two bacterial strains, more preferably at least one bacterial strain is a vitamin B12 autotroph.

[0124] 8. The method according to any one of items 1 to 7, wherein the at least one microbial strain comprises a microbial strain selected from Vibrio species, in particular Vibrio natriegens, Geobacillus species, in particular Geobacillus LC300, and Bacillus species, in particular Bacillus megaterium.

[0125] 9. The method according to any one of items 1 to 8, wherein at least one microbial strain comprises a microorganism capable of synthesizing omega-3 fatty acids.

[0126] 10. The method of any one of items 1 to 9, wherein at least one microbial strain comprises a genetically modified microorganism.

[0127] 11. The method according to any one of items 1 to 10, wherein step (b) involves a thermal pretreatment carried out at a temperature between 70°C and 180°C and / or for a time between 10 minutes and 360 minutes, followed by an enzymatic treatment with protease(s) preferably at a temperature between 40°C and 100°C, more preferably between 45°C and 70°C, and / or at a pH between 3.0 and 9.0, more preferably between 5.0 and 7.0, and / or for a period between 0.5 hours and 72 hours, more preferably between 0.5 hours and 10 hours.

[0128] 12. The method according to any one of items 1 to 10, wherein step (b) is carried out by mechanical homogenization, optionally with a thermal pretreatment carried out at a temperature between 70°C and 180°C and / or for a time between 10 minutes and 360 minutes.

[0129] 13. The method according to any one of items 1 to 12, wherein step (c) involves an enzymatic treatment with nuclease(s) preferably at a temperature of 40° C. to 100° C., more preferably 45° C. to 70° C., and / or at a pH of 3.0 to 9.0, more preferably 5.0 to 7.0, and / or for a period of 0.5 h to 72 h, more preferably 0.5 h to 10 h.

[0130] 14. The method according to any one of items 1 to 13, wherein step (c) further comprises an enzymatic treatment with deaminase(s) at a temperature of preferably 40° C. to 100° C., more preferably 45° C. to 70° C., and / or at a pH of 3.0 to 9.0, more preferably 5.0 to 7.0, and / or for a period of 0.5 hours to 72 hours, more preferably 0.5 hours to 10 hours.

[0131] 15. The method according to any one of items 1 to 14, wherein step (c) further comprises an enzymatic treatment with protease(s) at a temperature of preferably 40°C to 100°C, more preferably 45°C to 70°C, and / or at a pH of 3.0 to 9.0, more preferably 5.0 to 7.0, and / or for a period of 0.5 hours to 72 hours, more preferably 0.5 hours to 10 hours.

[0132] 16. A composition obtainable according to the method according to any one of items 1 to 15, comprising at least 22% nucleotides and at least 40% proteins and / or amino acids and peptides.

[0133] 17. The composition according to item 16, characterized by an IMP / GMP content of at least 11%, preferably at least 13%, even more preferably at least 15%, most preferably at least 17%.

[0134] 18. The composition according to item 16 or 17, further comprising a glutamic acid content of at least 6% (wt / wt), preferably at least 8% (wt / wt), more preferably at least 12% (wt / wt), even more preferably at least 14% (wt / wt), most preferably at least 16% (wt / wt).

[0135] 19. Use of the composition according to any one of items 16 to 18 in the manufacture of an animal feed, a pet food, a fermentation medium or supplement, a food, a dietary supplement, a pharmaceutical, a diagnostic agent, DNA or RNA, or a flavor enhancer.

[0136] 20. An animal feed, a pet food, a food, a fermentation medium or supplement, a nutritional supplement, a pharmaceutical, a diagnostic agent, DNA or RNA, or a flavor enhancer, comprising the composition according to any one of items 16 to 18.

[0137] 21. Use of a composition according to any one of items 16 to 18 as a replacement for monosodium glutamate or as / in an antibiotic replacement.

[0138] 22. Biomass obtainable in step (a) of the method according to any one of items 1 to 15.

[0139] 23. Biomass according to item 22, having a glutamic acid content of at least 5%, preferably at least 6% (wt / wt), more preferably at least 7% (wt / wt), even more preferably at least 8% (wt / wt), even more preferably at least 9% (wt / wt), even more preferably at least 10% (wt / wt), most preferably at least 12% (wt / wt).

[0140] 24. A single-cell protein comprising the biomass according to item 22 or 23.

[0141] 25. The single-cell protein according to item 24, further subjected to heat treatment.

[0142] 26. Use of the single-cell protein according to item 24 or 25 in the manufacture of an animal feed, a pet food or a food product, preferably an animal feed.

[0143] 27. A cell lysate obtainable in step (b) of the method according to any one of items 1 to 15.

[0144] 28. A composition comprising nucleotides obtainable by separation of a cell lysate according to item 27, preferably by ultrafiltration.

[0145] 29. A composition comprising amino acids and peptides obtainable by separation of a cell lysate according to item 27, preferably by ultrafiltration.

[0146] 30. Use of the composition according to item 28 or the composition according to item 29 in the manufacture of an animal feed, a pet food, a food product, a fermentation medium or supplement, a nutritional supplement, a pharmaceutical product, a diagnostic agent, a DNA or RNA, or a flavor enhancer.

[0147] 31. An animal feed, a pet food, a food product, a fermentation medium or supplement, a nutritional supplement, a pharmaceutical product, a diagnostic agent, a DNA or RNA, or a flavor enhancer, comprising the composition according to item 28 or the composition according to item 29.

[0148] Further examples and embodiments of the present invention are disclosed in the following numbered paragraphs.

[0149] 1. A method for producing a composition comprising at least 22% nucleotides and at least 40% amino acids and peptides, comprising: (a) At least 0.85 hours -1 Providing a biomass by culturing at least one microbial strain characterized by a growth rate during the process; (b) lysing the cells present in the biomass of step (a); (c) converting the nucleic acids present in the lysate of step (b) into nucleotides and, optionally, converting the proteins present in the lysate of (b) into amino acids and peptides; where % is understood to be % w / w of dry mass excluding NaCl.

[0150] 2. At least one microbial strain of step (a) is incubated for at least 1.1 h. -1 , preferably at least 1.2 h -1 , more preferably at least 1.4 h -1 and / or characterized by a growth rate during the process. at least one microbial strain of step (a) is characterized by a nucleic acid content of at least 22%, preferably at least 25%, even more preferably at least 28%; and / or Item 2. The method according to item 1, wherein the at least one microbial strain in step (a) comprises a bacterial strain, preferably a halophilic or thermophilic bacterium, preferably the bacterial strain is characterized by a genomic G / C content of at least 40%, more preferably at least 45%, even more preferably at least 50%, and most preferably at least 55%.

[0151] 3. At least one microbial strain in step (a) comprises a microorganism that is a vitamin B12 autotroph, or 3. The method according to item 1 or 2, wherein the at least one microbial strain in step (a) is two microbial strains, preferably two bacterial strains, more preferably at least one bacterial strain is a vitamin B12 autotroph.

[0152] 4. Step (a) is carried out for at least 0.85 h. -1 , preferably at least 1.1 h -1 , more preferably at least 1.2 h -1 , and even more preferably at least 1.4 h -1 The method according to any one of items 1 to 3, wherein the growth rate of the at least one microbial strain in each process of step (a) is limited by the dilution rate, and optionally, the at least one microbial strain is cultured under non-sterile conditions.

[0153] 5. The method according to any one of claims 1 to 4, wherein the at least one microbial strain comprises a microbial strain selected from Vibrio species, particularly Vibrio natriegens, Geobacillus species, particularly Geobacillus LC300, and Bacillus species, particularly Bacillus megaterium.

[0154] 6. At least one microbial strain comprises a microorganism capable of synthesizing omega-3 fatty acids; and / or 6. The method of any one of items 1 to 5, wherein at least one microbial strain comprises a genetically modified microorganism.

[0155] 7. step (b) involves a thermal pretreatment carried out at a temperature between 70°C and 180°C and / or for a time between 10 and 360 minutes, followed by an enzymatic treatment with protease(s) preferably at a temperature between 40°C and 100°C, more preferably between 45°C and 70°C, and / or at a pH between 3.0 and 9.0, more preferably between 5.0 and 7.0, and / or for a period between 0.5 and 72 hours, more preferably between 0.5 and 10 hours; and / or Step (b) is carried out by mechanical homogenization, optionally with a thermal pretreatment carried out at a temperature between 70°C and 180°C and / or for a time between 10 minutes and 360 minutes; and / or step (c) preferably involves an enzymatic treatment with nuclease(s) at a temperature of 40° C. to 100° C., more preferably 45° C. to 70° C., and / or at a pH of 3.0 to 9.0, more preferably 5.0 to 7.0, and / or for a period of 0.5 to 72 hours, more preferably 0.5 to 10 hours; Preferably, step (c) further comprises an enzymatic treatment with deaminase(s), preferably at a temperature of 40° C. to 100° C., more preferably 45° C. to 70° C., and / or at a pH of 3.0 to 9.0, more preferably 5.0 to 7.0, and / or for a period of 0.5 hours to 72 hours, more preferably 0.5 hours to 10 hours; The method according to any one of items 1 to 6, wherein step (c) preferably further comprises an enzymatic treatment with a protease(s) at a temperature of preferably 40° C. to 100° C., more preferably 45° C. to 70° C., and / or at a pH of 3.0 to 9.0, more preferably 5.0 to 7.0, and / or for a period of 0.5 hours to 72 hours, more preferably 0.5 hours to 10 hours.

[0156] 8. A method according to any one of items 1 to 7, comprising at least 22% nucleotides and at least 40% proteins and / or amino acids and peptides; Preferably, characterized by an IMP / GMP content of at least 11%, preferably at least 13%, even more preferably at least 15%, most preferably at least 17%. Preferably, the composition further comprises a glutamic acid content of at least 6% (wt / wt), preferably at least 8% (wt / wt), more preferably at least 12% (wt / wt), even more preferably at least 14% (wt / wt), most preferably at least 16% (wt / wt).

[0157] 9. Use of the composition according to item 8 in the manufacture of animal feed, pet food, fermentation medium or supplement, food, dietary supplement, pharmaceutical, diagnostic agent, DNA or RNA, or flavor enhancer.

[0158] 10. An animal feed, a pet food, a food product, a fermentation medium or supplement, a nutritional supplement, a pharmaceutical product, a diagnostic agent, a DNA or RNA, or a flavor enhancer, comprising the composition according to item 8.

[0159] 11. Use of the composition according to paragraph 8 as a replacement for monosodium glutamate or as / in an antibiotic replacement.

[0160] 12. Biomass obtainable in step (a) of the method according to any one of items 1 to 7, preferably having a glutamic acid content of at least 5%, more preferably at least 6% (wt / wt), even more preferably at least 7% (wt / wt), even more preferably at least 8% (wt / wt), even more preferably at least 9% (wt / wt), even more preferably at least 10% (wt / wt), most preferably at least 12% (wt / wt).

[0161] 13. A single-cell protein comprising biomass according to item 12, preferably wherein the biomass has undergone heat treatment.

[0162] 14. Use of the single-cell protein according to item 13 in the manufacture of animal feed, pet food or food, preferably animal feed.

[0163] 15. A cell lysate obtainable in step (b) of the method according to any one of items 1 to 7.

[0164] 16. A composition comprising nucleotides obtainable by separation of a cell lysate according to item 15, preferably by ultrafiltration.

[0165] 17. A composition comprising amino acids and peptides obtainable by separation of a cell lysate according to item 15, preferably by ultrafiltration.

[0166] 18. Use of the composition according to item 16 or the composition according to item 17 in the manufacture of animal feed, pet food, food, fermentation medium or supplement, nutritional supplement, pharmaceutical, diagnostic agent, DNA or RNA, or flavor enhancer.

[0167] 19. An animal feed, a pet food, a food product, a fermentation medium or supplement, a nutritional supplement, a pharmaceutical product, a diagnostic agent, a DNA or RNA, or a flavor enhancer, comprising the composition according to item 16 or the composition according to item 17.

[0168] Various modifications and variations of the present invention will become apparent to those skilled in the art without departing from the scope of the present invention. Although the present invention has been described in terms of certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be covered by the present invention.

[0169] The present invention is illustrated by the following examples which are not intended to be construed as limiting. EXAMPLES

[0170] General Experimental Protocol I. Fermentation conditions To produce biomass from V. natriegens that was later used to produce the extract, 3 × 100 mL preculture flasks were prepared using a method derived from Harwood and Cutting (Harwood and Cutting, 1990, Molecular Biological Methods for Bacillus. New York, NY: Wiley) and diluted with 5 g L -1 sugar or glucose from beet molasses, 1g L -1 NH4Cl, 3 g L -1 KH2PO4, 0.5 g L -1 NaCl, 6.7 g L -1 Na2HPO4, 1 mg L -1 MnCl2 4H2O, 1.7 mg L -1 ZnCl2, 430 μg L -1 CuCl2 2H2O, 328 μg L -1 CoCl2, 600 μg L -1 NaMoO4 2H2O, 11.1 mg L -1 CaCl2, 30 mg L -1 of 3,4-dihydroxybenzoic acid (3,4-DHB), 13.5 mg L -1 of FeCl3, and 120 mg L -1The culture was prepared with 5 mL of modified M9 medium containing 1000 ng / ml MgSO4. The culture was incubated overnight at 37°C and 270 rpm, then collected in a 15 mL falcon tube and subsequently centrifuged (10 min, 4500 rpm, 4°C). The supernatant was removed, the pellet was washed with sterile 0.9% NaCl, and then the resuspended cell suspension was used to inoculate a 15 L fermenter containing the same medium with a working volume of 10 L. The fermenter was operated at a constant temperature of 37°C and the pH was adjusted to 7 during the fermentation process using 25% ammonia. Similarly, the aeration rate and the agitation rate were set at 1.5 VVM and 400 rpm, respectively, at the beginning, but were then increased to keep the dissolved oxygen (DO) at 30% throughout the entire course of the fermentation. After fermentation, the medium was directly cooled to 10°C-15°C, and the biomass was separated by centrifugation (4500 rpm, 10 min, 4°C), washed with 0.9% NaCl, and centrifuged again. The biomass was then stored at 4°C before further processing.

[0171] II. Autolysis For autolysis, the biomass obtained was diluted with water to obtain a suspension with a dry mass of exactly 16%. 100 mL of this suspension was then incubated in a water bath at 49°C and 150 rpm for 14 hours, after which the temperature was raised to 56°C for an additional 6 hours of autolysis. The resulting solution was then centrifuged to remove cell wall debris and the supernatant (produced extract) was transferred to a tube and lyophilized to obtain a powder. The composition of the extract was analyzed according to the methods described herein.

[0172] III. Mechanical lysis followed by enzymatic treatment Mechanical lysis and subsequent enzymatic treatment are preferably performed as follows, unless otherwise indicated. For mechanical lysis, the biomass was resuspended in water to obtain a final dry mass of 9%-10% and then homogenized at 1500 bar. Throughout the process, the suspension and homogenizer were kept at 4 °C to ensure the stability of the nucleic acids. After homogenization, a part of the suspension was freeze-dried to analyze the resulting extract. Another part of the extract was subjected to various enzymatic treatments to convert the proteins and nucleic acids from the previous extract into amino acids, peptides, and nucleotides. For that, 0.4% Nuclease E7 from Amano Enzyme Inc. (Japan) was added to the suspension, which was then incubated at 65 °C for 16 h. Then, 0.2% Protease M was added and the suspension was incubated at 50 °C to convert the proteins into amino acids.

[0173] Alternatively, mechanical lysis followed by enzymatic treatment may be performed as follows. For mechanical lysis, the biomass was resuspended in water to obtain a final dry mass of 19%-20% and then homogenized at 1500 bar. Throughout the process, the suspension and homogenizer were kept at 4 °C to ensure the stability of the nucleic acids. After homogenization, the cell walls were discarded by centrifugation (4500 rpm, 10 min, 4 °C) and a portion of the supernatant was freeze-dried to analyze the resulting extract. Another portion of the extract was subjected to various enzymatic treatments to convert proteins and nucleic acids from the previous extract into amino acids, peptides, and nucleotides. For that, 0.1% (wt / wt) Deamizyme T, 0.2% Nuclease E7, and 0.05% YL-T L from Amano Enzyme Co., Ltd. (Japan) were added to the suspension, which was then incubated at 65 °C for 4 h. Then, 0.1% Prote AXH was added and the suspension was incubated at 50 °C to convert proteins into amino acids.

[0174] Analysis method I. Protein content determination First, the nitrogen content in the samples was determined using the Kjeldahl method (ASU method L 06.00-7 (2014 / 08)), then multiplied by the factor 6.25 typically applied in the food industry, and finally converted to protein content by subtracting the nucleic acid content, since it also contains nitrogen. The total protein content could also be confirmed by analysis of the digested amino acid content.

[0175] II. Analysis of Amino Acid Content The extracts were spiked with α-aminobutyric acid to a final concentration of 200 μM, allowing the use of α-aminobutyric acid as an internal standard. The amino acid concentrations in the prepared samples were finally measured by fluorescence detection using an Agilent 1200 HPLC system (Agilent technologies, Waldbronn, Germany) equipped with a reversed-phase column Gemini 5μ C18 110 A (150 × 4.6 mm, Phenomenex, Aschaffenburg, Germany) as stationary phase. The separation of the amino acids constituting the different proteins relied on a gradual change in the mobile phase composition throughout the run by separately mixing eluent A (40 mM NaH2PO4, pH 7.8) and eluent B (45% methanol, 45% acetonitrile, 10% water) according to a well-defined gradient profile. Furthermore, the column separation was performed at 40 °C with a flow rate of 1 mL min−1. -1 In addition, a precolumn (Gemini C18, MAX, RP, 4 × 3 mm, Phenomenex, Aschaffenburg, Germany) was used to extend the column life. Fluorescence detection was achieved by precolumn derivatization with o-phthalaldehyde (OPA) and 9-fluorenylmethyloxycarbonyl (FMOC) and by changing the excitation and emission wavelengths (Table 1).

[0176] [Table 1]

[0177] III. Analysis of Ribonucleic Acid (RNA) Content To measure nucleic acid content, a protocol adapted from Benthin et al. (1991) was used. Briefly, cells were first washed with 700 mM HClO4, then incubated with 300 mM KOH at 37°C for 300 min. -1 The cells were digested with 500 mM HClO4 for 80 min. After lysis, the resulting suspension was cooled and neutralized with 3 M HClO4. The supernatant was then collected by centrifugation (4500 rpm, 4°C, 10 min) and the remaining cell debris was washed again with 500 mM HClO4 to recover the remaining nucleic acids and to remove precipitated KClO4. All supernatants were mixed and the nucleic acid content of the final solution was finally quantified by spectrophotometry with a NanoDrop 1000™ (Thermo Fisher Scientific, Waltham, MA, USA).

[0178] IV. Determination of Deoxyribonucleic Acid (DNA) Content Concentration To disrupt the cell walls, the cell pellet was lysed in 560 μL of DNA lysis buffer (see Table 2) in a 2 mL tube at 30 °C for 350 min. -1 The cells were incubated at 4°C for 30 min and then subjected to mechanical lysis with glass beads (20% (vol / vol), 0.038 mm to 0.045 mm) in a FastPrep-24™ to ensure complete cell lysis. The resulting extracts were then incubated at 4°C for 13,200 min. -1 Centrifuge for 5 min at 60 µg L and collect the supernatant containing cytosolic compounds. -1 The ribonucleic acids in the samples were completely digested by treatment with 140 μL of a solution containing RNase. Afterwards, DNA from the samples was purified using a first separation step with 700 μL of Roti-phenol-chloroform-isoamyl alcohol, followed by a second separation step with 700 μL of chloroform. The supernatant obtained after centrifugation was then supplemented with 65 μL of 3 M sodium acetate (pH 5.5) and ice-cold pure DNA to induce DNA precipitation. After centrifugation, the supernatant was discarded and the resulting DNA pellet was washed with 70% ethanol. Finally, the ethanol was removed by evaporation and the DNA pellet was resuspended in 100 μL of ultrapure water before the concentration was measured using Nanodrop 1000™.

[0179] [Table 2]

[0180] V. Determination of Nucleotide Concentration The nucleotide content (preferably understood herein as the content of nucleotides, nucleosides and nucleic acid bases, more preferably the content of nucleotides and nucleosides) in the produced extracts was determined by ion-pair reversed-phase high performance liquid chromatography (IPRP-LC) using an Agilent 1100 system followed by diode array detection (DAD) at 254 nm. Separation was carried out using a Synergy Hydrocolumn as stationary phase and a mobile phase consisting of a mixture of 50 mM phosphate buffer (pH 5.8) and methanol at a flow rate of 0.4 mL / min and a temperature of 20°C. To achieve a proper separation of the analytes, the composition of the mobile phase was changed during the run according to the following gradient: 50% methanol from 0 min to 3 min, 0% to 50% methanol from 3 min to 12 min and finally 50% methanol from 12 min to 13.5 min.

[0181] Example 1 - Growth of V. natriegens biomass V. natriegens biomass was prepared by growth on molasses or glucose as a carbon source as described herein, and the growth curves are shown in FIG.

[0182] Example 2 - Composition of V. natriegens biomass The biomass from Example 1 grown on molasses was used to determine the total protein content, amino acid profile, and DNA and RNA content using the methods described above. The results are summarized in Table 3. 90% of all available nucleic acids (RNA and DNA) was RNA.

[0183] [Table 3]

[0184] Further information regarding the composition, i.e., amino acid profile, of the resulting biomass is contained in Table 4.

[0185] [Table 4]

[0186] Example 3 - V. Composition of autolysate from Natriegens The biomass of Example 1 grown on molasses was subjected to autolysis as defined herein. The total protein content, amino acid profile and RNA content were determined using the methods described above.

[0187] The obtained autolysate contained 2% (wt / wt) of nucleotides, 3% of nucleosides, and 1% of nucleic acid bases. RNA was also present at 3%. This gave a total of 8% (wt / wt) of nucleic acids and their derivatives. The total protein content, taken as the sum of all available amino acids, was 54% (wt / wt). Information on the amino acid profile of the obtained autolysate is contained in Table 5.

[0188] [Table 5]

[0189] Example 4 - Composition of single cell proteins The biomass of Example 1 grown on molasses was subjected to heat treatment where it was inactivated and further dried. The total protein content, amino acid profile and RNA content were determined using the methods described above. The nucleotide content was not determined since no lysis or enzymatic hydrolysis steps were performed for the production of single cell proteins.

[0190] The single-cell protein obtained contained 25% RNA (wt / wt, dry weight excluding NaCl). The total protein content, obtained as the sum of all available amino acids, is 55% (wt / wt, dry weight excluding NaCl). Information on the amino acid profile of the obtained single-cell protein is included in Table 6. The glutamic acid content for the single-cell protein was 8% (wt / wt, dry weight excluding NaCl).

[0191] [Table 6]

[0192] Example 5 - Composition of the lysate after mechanical homogenization The molasses-grown biomass of Example 1 was subjected to mechanical homogenization as defined herein.

[0193] The resulting homogenate contained 23% RNA (wt / wt, dry weight excluding NaCl) as determined using the method described above. The total content of nucleotides, nucleosides, and nucleobases in the solution was 0.5% (wt / wt, dry weight excluding NaCl).

[0194] Example 6 - Composition of enzyme extracts prepared from lysates obtained from mechanical homogenization The molasses-grown biomass of Example 1 was mechanically homogenized and subsequently subjected to further enzymatic treatment as defined above. The total protein content, amino acid profile, and RNA content were determined using the methods described above.

[0195] The enzyme extract contained 15% (wt / wt, dry weight excluding NaCl) nucleotides, 3% nucleosides, and 0.2% nucleobases, as defined herein, for a total of 18% (wt / wt, dry weight excluding NaCl) free nucleotides. There was still 6% RNA remaining in the suspension, which means that the total of nucleic acids and their derivatives was about 24% (wt / wt, dry weight excluding NaCl).

[0196] The total protein content, taken as the sum of all available amino acids, was 53% (w / w). Further information on the amino acid profile of the obtained extract is contained in Table 7. The glutamic acid content for the enzyme extract was 7% (w / w, dry weight excluding NaCl).

[0197] [Table 7]

Claims

1. A method for producing a composition comprising at least 15% nucleotides and at least 40% amino acids and peptides, comprising: (a) at least 0.85 h －１ providing biomass by culturing at least one microbial strain characterized by a growth rate during a process of (b) lysing the cells present in the biomass of step (a); (c) converting the nucleic acids present in the lysate of step (b) into nucleotides; wherein %, is understood as weight / weight % of the dry mass excluding NaCl.

2. The method according to claim 1, wherein step (c) further comprises converting the proteins present in the lysate of step (b) into amino acids and peptides.

3. The method according to claim 1, wherein the composition comprising at least 15% nucleotides and at least 40% amino acids and peptides is a composition comprising at least 18% nucleotides and at least 40% amino acids and peptides.

4. The method according to claim 1, wherein the composition comprising at least 15% nucleotides and at least 40% amino acids and peptides is a composition comprising at least 22% nucleotides and at least 40% amino acids and peptides.

5. The at least one microbial strain of step (a) is characterized by a growth rate during the process of at least 1.1 h-1, and / or The at least one microbial strain of step (a) is characterized by a nucleic acid content of at least 15%, according to the method of claim 1.

6. The at least one microbial strain of step (a) comprises a bacterial strain, the at least one microbial strain of step (a) comprises a halophilic or thermophilic bacterium, or the at least one microbial strain of step (a) comprises a bacterial strain characterized by a genomic G / C content of at least 40%; and / or The at least one microbial strain of step (a) comprises a microorganism that is a vitamin B12 auxotroph, according to the method of claim 1.

7. Step (a) is carried out as a continuous process at a dilution rate of at least 0.85 h-1, and the growth rate of each of the at least one microbial strain of step (a) in the process is limited by the dilution rate, according to the method of claim 1.

8. The method according to claim 7, wherein the at least one microbial strain is cultured under non-sterile conditions.

9. The method according to claim 1, wherein the at least one microbial strain of step (a) is two bacterial strains.

10. The method according to claim 1, wherein the at least one microbial strain comprises a microbial strain selected from Vibrio species, in particular Vibrio natriegens, Geobacillus species, in particular Geobacillus LC300, and Bacillus species, in particular Bacillus megaterium.

11. The method according to claim 1, wherein the at least one microbial strain comprises a microorganism capable of synthesizing omega-3 fatty acids.

12. The method according to claim 1, wherein the at least one microbial strain comprises a genetically modified microorganism.

13. Step (b) involves a heat pretreatment carried out at a temperature of 70°C to 180°C and / or for a time of 10 minutes to 360 minutes, followed by an enzymatic treatment with at least one protease, or Step (b) is carried out by mechanical homogenization, optionally accompanied by a heat pretreatment carried out at a temperature of 70°C to 180°C and / or for a time of 10 minutes to 360 minutes; and / or Step (c) involves an enzymatic treatment with at least one nuclease; and / or Step (c) further comprises an enzymatic treatment with at least one deaminase, or The method according to claim 1, wherein step (c) further comprises an enzymatic treatment with at least one protease.

14. A composition comprising at least 15% nucleotides, and at least 40% proteins and / or amino acids and peptides, obtainable by the method according to any one of claims 1 to 13.

15. The composition according to claim 14, comprising at least 18% nucleotides.

16. The composition according to claim 14, comprising at least 22% nucleotides.

17. The composition according to claim 14, characterized by an IMP / GMP content of at least 11%.

18. The composition according to claim 14, further comprising a glutamic acid content of at least 6% (weight / weight).

19. The composition according to claim 18, comprising a glutamic acid content of at least 8% (weight / weight).

20. Use of the composition according to claim 14 in the manufacture of animal feed, pet food, fermentation media or supplements, food, dietary supplements, pharmaceuticals, diagnostics, DNA or RNA, or flavor enhancers.

21. Use of the composition according to claim 18 in the manufacture of animal feed, pet food, fermentation media or supplements, food, dietary supplements, pharmaceuticals, diagnostics, DNA or RNA, or flavor enhancers.

22. An animal feed, pet food, food, fermentation medium or supplement, dietary supplement, pharmaceutical, diagnostic, DNA or RNA, or flavor enhancer comprising the composition according to claim 14.

23. An animal feed, pet food, food, fermentation medium or supplement, dietary supplement, pharmaceutical, diagnostic, DNA or RNA, or flavor enhancer comprising the composition according to claim 18.

24. Use of the composition according to claim 14 as a sodium glutamate substitute or as / in an antibiotic substitute.

25. Use of the composition according to claim 18 as a sodium glutamate substitute or as / in an antibiotic substitute.

26. Biomass obtainable in step (a) of the method according to any one of claims 1 to 13.

27. The biomass according to claim 26, wherein the glutamic acid content is at least 5% (weight / weight).

28. Single cell protein comprising the biomass according to claim 26.

29. The single cell protein according to claim 28, wherein the at least one microbial strain is Vibrio natriegens.

30. Single cell protein comprising the biomass according to claim 27.

31. The single cell protein according to claim 30, wherein the at least one microbial strain is Vibrio natriegens.

32. The single cell protein according to claim 28, further subjected to heat treatment.

33. The single cell protein according to claim 29, further subjected to heat treatment.

34. Use of the single cell protein according to claim 28 in the manufacture of animal feed, pet food or food.

35. Use of the single cell protein according to claim 29 in the manufacture of animal feed, pet food or food.

36. Cell lysate obtainable by step (b) of the method according to any one of claims 1 to 13.

37. A composition comprising nucleotides obtainable by separation of the cell lysate according to claim 36.

38. A composition comprising amino acids and peptides obtainable by separation of the cell lysate according to claim 36.

39. Use of the composition according to claim 37 in the manufacture of animal feed, pet food, food, fermentation medium or supplement, dietary supplement, pharmaceutical, diagnostic agent, DNA or RNA, or flavor enhancer.

40. Use of the composition according to claim 38 in the manufacture of animal feed, pet food, food, fermentation medium or supplement, dietary supplement, pharmaceutical, diagnostic agent, DNA or RNA, or flavor enhancer.

41. Animal feed, pet food, food, fermentation medium or supplement, dietary supplement, pharmaceutical, diagnostic agent, DNA or RNA, or flavor enhancer comprising the composition according to claim 37.

42. Animal feed, pet food, food, fermentation medium or supplement, dietary supplement, pharmaceutical, diagnostic agent, DNA or RNA, or flavor enhancer comprising the composition according to claim 38.

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