Attenuation of lipopolysaccharide-induced toxicity in bacterial biomass

Abstract

A process for providing a biomass fraction from a bacterial biomass that has reduced toxicity to the bacterial biomass is described. Also described is a bacterial biomass fraction having a lipopolysaccharide (LPS) content of less than 5% w / w of the bacterial biomass fraction. Also provided is an animal feed comprising or consisting of the bacterial biomass fraction.

Description

[Technical Field]

[0001] The present invention relates to a process for providing a biomass fraction having reduced toxicity from a bacterial biomass. Further, the present invention relates to a bacterial biomass fraction having a lipopolysaccharide content of less than 5% w / w of the bacterial biomass fraction. [Background technology]

[0002] The Gram-negative methanotroph Methylococcus capsulatus is a non-symbiotic bacterium found widely in nature. It metabolizes methane, for example from natural gas, into biomass, CO2, and water. Due to its high protein content, M. capsulatus can be used as a protein supplement in animal feed and is also of interest for human consumption. Fermentation of this bacterium as a protein source for both animal and human consumption could contribute to meeting the world's need for dietary protein in a more environmentally friendly way than traditional protein production industries.

[0003] Bacterial lipopolysaccharide (LPS, also known as endotoxin) is a bacterial toxin that has been linked to many diseases, including liver damage, neurodegeneration, chronic intestinal inflammation, and diabetes, among others. LPS naturally occurs in bacterial cells and is their structural component. LPS consists of long chains of polysaccharides covalently linked to lipids. Bacterial cell membranes are composed of a bilayer lipid layer interspersed with proteins. The outer layer is LPS, which contains the phosphorylated lipid lipid, lipid A. The toxicity of LPS is thought to be primarily due to lipid A, while the polysaccharide portion of LPS is generally considered to be less toxic.

[0004] LPS acts as a pyrogenic compound, causing fever when injected directly into animals, and is lethal in both humans and animals when injected intravenously at 1-2 micrograms. Wassenaar et al. (Wassenaar TM, Zimmermann K. Eur J Microbiol Immunol (Bp). 2018 Aug 21;8(3):63-69. doi:10.1556 / 1886.2018.00017) report on LPS and its toxicity when ingested or injected into animals or humans. LPS removal from biomass is described in FI129784.

[0005] As a Gram-negative organism, M. capsulatus naturally has a high content of lipopolysaccharide (LPS). This poses a potential problem in the production and processing of bacterial biomass derived from Gram-negative organisms. Inhalation of dust from such processes can allow LPS to enter the bloodstream, potentially causing liver damage in the long term.

[0006] It is therefore an object of embodiments of the present invention to provide a bacterial biomass with reduced toxicity, as well as a process for its production. Safety in handling and storage of such products can therefore be improved. Summary of the Invention

[0007] By subjecting bacterial biomass to various process steps, the inventors have found that the resulting product is less harmful in that it contains reduced concentrations of bacterial outer cell membrane components.

[0008] Thus, in a first aspect, the present invention relates to a process for providing a biomass fraction having reduced toxicity to a bacterial biomass, said process comprising the steps of: 1a. suspending a bacterial biomass in a solution comprising a chelating agent to provide a first cell suspension; 1b. incubating the first cell suspension for a predetermined period of time; and 1c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, whereby the first biomass fraction has reduced toxicity compared to said bacterial biomass.

[0009] In a second aspect, the present invention relates to a process for providing a biomass fraction from a bacterial biomass having reduced toxicity to said bacterial biomass, said process comprising: 2a. lysing at least a portion of bacterial cells in said bacterial biomass to provide a second biomass fraction; and 2b. incubating the second biomass fraction for a predetermined period of time, whereby the second biomass fraction has reduced toxicity compared to said bacterial biomass.

[0010] In a third aspect, the present invention provides a process for providing a biomass fraction from a bacterial biomass that has reduced toxicity to the bacterial biomass, the process combining the process steps of the first and second aspects. In this third process, the process comprises the steps of: 3a. suspending a bacterial biomass in a solution comprising a chelating agent to provide a first cell suspension; 3b. incubating the first cell suspension for a predetermined period of time; 3c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction; and 3d. separating the Ca chelating agent from the first cell suspension. 2+ , Mg 2+ , Cu 2+ , Fe 2+ , Co 2+ , Al 2+3e. lysing at least a portion of the bacterial cells in said first biomass fraction, wherein steps 3d and 3e may be performed in either order to provide a third biomass fraction; 3f. incubating the third biomass fraction for a predetermined period of time, whereby the third biomass fraction has reduced toxicity compared to said bacterial biomass.

[0011] In a fourth aspect, the present invention relates to a bacterial biomass fraction having a lipopolysaccharide (LPS) content of less than 5% w / w, more preferably less than 2% w / w, even more preferably less than 1% w / w of the bacterial biomass fraction.

[0012] In a fifth aspect, the present invention relates to an animal feed comprising or consisting of a bacterial biomass fraction according to the fourth aspect.

[0013] In a sixth aspect, the present invention relates to a human food comprising or consisting of a bacterial biomass fraction according to the fourth aspect. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is an overview of the first process according to the present invention. [Figure 2] FIG. 2 is an overview of a second process according to the present invention. [Figure 3] FIG. 3 is an overview of a third process according to the present invention, where process steps 3a-3e are shown to include optional washing and centrifugation steps.

[0015] definition The term "biomass" refers to proteinaceous products, which may be in the form of protein extracts, including cell wall material of single-celled microorganisms from pure or mixed cultures of algae, yeast, fungi, or bacteria.

[0016] The term "biomass fraction" refers to a fraction of biomass, i.e., a portion of biomass.

[0017] The term "single cell protein (SCP)" generally refers to a proteinaceous product isolated from a single-celled microorganism. The proteinaceous product may be in the form of biomass or protein extracts and includes cell wall material of single-celled microorganisms from pure or mixed cultures of algae, yeast, fungi, or bacteria. Single-cell proteins have traditionally been used as sources or substitutes for protein-rich foods and are suitable for human consumption or animal feed.

[0018] The use of microorganisms to obtain biomass for use in feed and food results in a product with a higher percentage of nucleic acids than conventional foods. The concentration of nucleic acids present in SCP varies depending on the specific microorganism used, but typically about 5-18% nucleic acids (dry weight) are present in SCP.

[0019] Throughout this text, the abbreviation "DM" means "Dry Matter." In this context, the terms "dry matter" and "ash" content are determined according to AOAC methods (Reference AOAC Standards, 1945).

[0020] As used herein, the term "dry weight," in the context of the dry weight of M. capsulatus biomass, should be taken to mean the weight of the biomass after all water has been removed from the biomass. This should not be taken to mean that in all embodiments of M. capsulatus biomass according to the present invention, all water has been removed, although in some embodiments water is present. Rather, it should be understood as a measure that can be used to reproducibly calculate whether a biomass falls within the range of biomass according to the present invention.

[0021] The term "reduced toxicity" refers to reduced toxicity of a biomass fraction produced by a microorganism, such as a pure or mixed culture of algae, yeast, fungi, or bacteria, thereby improving the safety of handling and storage of the biomass fraction. This may be, for example, reducing the LPS content in the biomass fraction; thus, reduced toxicity may refer to a reduced LPS content in the biomass fraction compared to untreated bacterial biomass.

[0022] The term "solution containing a chelating agent" refers to a solution containing chelating agent molecules, i.e., molecules capable of binding to metal ions. Chelation typically involves the formation of two or more separate coordinate bonds between a multidentate (multiple-binding) ligand and a single metal ion.

[0023] As previously mentioned, the present invention provides various processes for providing a biomass fraction from a bacterial biomass that has reduced toxicity relative to the bacterial biomass.

[0024] fermentation The biomass material (which is typically aqueous biomass material) is suitably obtained from the fermentation of at least one microorganism, preferably at least one of which is a bacterial cell, preferably a methanotrophic bacterium, more preferably M. capsulatus.

[0025] In the fermentation step, a microorganism, or a mixture of microorganisms, produces methane from aqueous biomass material and CO. 2, and water. Fermentation is carried out in a fermentation tank, the process of which is described in detail, for example, in WO2017 / 080987 and WO2022 / 008478, which are incorporated herein by reference.

[0026] Biomass material is a single-cell protein (SCP) product. It contains primarily protein (approximately 60%) with small amounts of RNA and DNA. When isolated from the fermentation step, the biomass material is an aqueous suspension. In this aqueous suspension, the majority of the solid components are cellular material from the microorganisms. Other components (e.g., proteins, nucleic acids, polysaccharides, lipids, LPS, or other small molecules) may be dissolved or suspended in the aqueous phase.

[0027] At least one of the microorganisms used in the fermentation step is suitably a bacterial cell, preferably a Gram-negative bacterial cell, preferably a methanotrophic bacterium, more preferably M. capsulatus, and therefore the biomass is suitably M. capsulatus biomass.

[0028] As used herein, the terms "Methylococcus capsulatus" or "M. capsulatus" refer to any strain of bacteria belonging to the M. capsulatus species. The strain may be naturally occurring or developed in a laboratory, such as a genetically modified strain. The term "naturally occurring" means that the strain has not been genetically modified using genetic engineering techniques. However, it may contain natural modifications or variations in its genetic material compared to a reference strain, such as variations that occur randomly during replication. Preferably, the strain is naturally occurring. Also preferably, the strain is M. capsulatus (Bath), more preferably M. capsulatus (Bath) identified as NCIMB 11132. However, it may also be M. capsulatus (Texas), or M. capsulatus (Aberdeen), or a different M. capsulatus strain currently known or to be discovered or characterized in the future.

[0029] Methanotrophic bacteria can be used in co-fermentation with one or more heterotrophic bacteria. The following heterotrophic bacteria can be particularly useful in co-fermentation with M. capsulatus: Ralstonia sp.; Bacillus brevis; Brevibacillus agri; Alcaligenes acidovorans; Aneurinibacillus danicus; and Bacillus firmus. Suitable yeasts can be selected from Saccharomyces and / or Candida species. Preferred heterotrophic bacteria are selected from Alcaligenes acidovorans (NCIMB 13287), Aneurinibacillus danicus (NCIMB 13288), and Bacillus firmus (NCIMB 13289), and combinations thereof. The methanotrophic and / or heterotrophic bacteria may be genetically modified.

[0030] In the fermentation step, the carbon source is converted into biomass material by microorganisms. Preferably, the carbon source comprises methane, such as natural gas, synthetic gas, or biogas. During the fermentation step, the carbon source is dissolved in a fermentation medium. Fermentation is preferably carried out in a U-loop reactor, as described in WO2010 / 069313, which is incorporated herein by reference. Suitable fermentation media are, for example, described in WO2018 / 158322, which is incorporated herein by reference. The fermentation step has a relatively low dryness, for example, less than 5%.

[0031] Co-fermentation of M. capsulatus with one or more other organisms can result in a biomass product that includes M. capsulatus biomass as well as the biomass of one or more other organisms.

[0032] In some embodiments, M. capsulatus is fermented in combination with one or more bacteria selected from: Ralstonia sp., B. brevis, B. agri, A. acidovorans, A. danicus, and B. firmus; preferably any one, two, or all three of: A. acidovorans, A. danicus, and B. firmus; more preferably any one, two, or all three of: A. acidovorans (NCIMB 13287), A. danicus (NCIMB 13288), and B. firmus (NCIMB 13289).

[0033] Further details of the fermentation process are described in WO2020 / 245197 and WO2020 / 249670, which are incorporated herein by reference.

[0034] The dry matter content of the biomass material from the fermentation process is between 1 and 3%. After recovery, the biomass material may be clarified, and the supernatant from the clarification step may be recycled to the fermenter. This results in pellets with a dry matter content of 10 to 18%. The fermentation broth in the fermenter is preferably continuously fed with the necessary amounts of water and nutrients, such as sulfate, chloride, or nitrate, ammonium / ammonia in the form of phosphate, magnesium, calcium, potassium, iron, copper, zinc, manganese, nickel, cobalt, and molybdenum, and pH control components commonly used by those skilled in the art, i.e., acids and / or bases, such as sulfuric acid (H2SO4), nitric acid (HNO3), sodium hydroxide (NaOH), and potassium nitrate (KNO3). The latter is also a suitable nitrogen source for M. capsulatus. Specific details of the fermentation process, including suitable substrates, are described in WO2000 / 70014 and WO2010 / 069313, which are incorporated by reference.

[0035] Biomass material produced from the fermentation of natural gas typically contains 60-80% crude protein by weight, 5-20% crude fat by weight, 3-12% ash by weight, and 3-15% nucleic acids (RNA and DNA).

[0036] Optionally, the biomass material is subjected to a diafiltration step at this point to reduce the dry matter content to about 6%.

[0037] A first process is a process for providing a biomass fraction from a bacterial biomass that has reduced toxicity to said bacterial biomass, said process comprising: 1a. suspending a bacterial biomass in a solution comprising a chelating agent to provide a first cell suspension; 1b. Incubating the first cell suspension for a predetermined period of time; 1c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction; The process involves a first biomass fraction having reduced toxicity compared to said bacterial biomass.

[0038] Figure 1 shows the steps of the first process.

[0039] suspension In a first step of the first process, a bacterial biomass is suspended in a solution containing a chelating agent to provide a first cell suspension. The bacterial biomass is derived from the fermentation of at least one methanotrophic bacterium, preferably M. capsulatus.

[0040] Prior to this step, the process may further comprise subjecting the bacterial biomass to an initial separation step to remove a first liquid fraction.

[0041] The solution containing the chelating agent and / or the chelating agent is a metal ion chelating agent, preferably a divalent metal ion chelating agent, preferably EDTA, ethylenediamine, glycine, citric acid, gluconic acid, tartaric acid, hexametaphosphate, pyrophosphate, tripolyphosphate, phytic acid, free histidine, L-glutamic acid, N,N-diacetic acid, aspartic acid, citrate, gluconate, or a salt thereof, in particular a sodium salt thereof, such as sodium citrate (TSA), sodium gluconate, preferably sodium EDTA, EDTA, sodium citrate (TSA), sodium gluconate, or a mixture thereof.

[0042] Without being bound by theory, metal chelators may cause destabilization of the outer cell membrane, reducing nucleic acid in the bacterial biomass. Sequestration of divalent cations is known to destabilize the outer membrane; see, e.g., THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 243, No. 24, Issue of December 25, pp. 6384-491, 1968, and FEMS Microbiology Letters 117 (1994) 203-206.

[0043] incubation In the second step, the first cell suspension is incubated for a predetermined time. The incubation of the first cell suspension is carried out under one or more of the following conditions: a pH between 5 and 11, preferably between 7 and 9, more preferably between 7.5 and 8.5; a temperature between 5 and 90°C, preferably between 20 and 90°C, more preferably between 30 and 90°C; a time period between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minute and 60 minutes, and more preferably between 1 minute and 30 minutes; 10 8 From 10 14 cells / mL, preferably between 10 10 From 10 12 The concentration of cells in the first cell suspension between 100 and 200 cells / mL.

[0044] separation In a third step, the incubated first cell suspension is subjected to a separation step to provide a liquid fraction and a first biomass fraction, whereby the first biomass fraction has reduced toxicity compared to the bacterial biomass.

[0045] One or more steps of washing and centrifugation of the first biomass fraction may be performed after the third step.

[0046] Combining the chelation and heating (incubation) steps will maximize / promote shedding of the outer membrane of Gram-negative bacteria, thus shedding endotoxins and simultaneously reducing nucleic acids in the bacterial biomass product.

[0047] A second process is a process for providing a biomass fraction from a bacterial biomass that has reduced toxicity to said bacterial biomass, said process comprising: 2a. Providing a second biomass fraction by lysis of at least a portion of the bacterial cells in said bacterial biomass; 2b. incubating the second biomass fraction for a predetermined period of time; The process involves a second biomass fraction having reduced toxicity compared to the bacterial biomass.

[0048] Figure 2 shows the steps of the first process.

[0049] Dissolution In a first step of the second process, lysis of at least a portion of the bacterial cells in the bacterial biomass is carried out to provide a second biomass fraction.

[0050] The lysis step comprises one or more steps selected from heating, cooling, freeze-thawing, sonication, mechanical treatment such as homogenization, chemical treatment, enzymatic treatment, pressure application, and filtration, preferably heating.

[0051] The homogenization parameters are 800 bar and a flow rate of 80-100 liters / hour. This step is preferably followed by UHT treatment.

[0052] incubation In a second step of the second process, the second biomass fraction is cultured for a predetermined time, whereby the second biomass fraction has reduced toxicity compared to the bacterial biomass.

[0053] Incubation of the second biomass fraction is carried out under one or more of the following conditions: a pH between 5 and 11, preferably between 7 and 9, more preferably between 7.5 and 8.5; a temperature between 5 and 90°C, preferably between 20 and 60°C, more preferably between 30 and 50°C; a time period between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minute and 60 minutes, and more preferably between 1 minute and 30 minutes; 10 8 From 10 14 cells / mL, preferably between 10 10 From 10 12 The concentration of cells in the first cell suspension between 100 and 200 cells / mL.

[0054] The third process relates to a process for providing a biomass fraction from a bacterial biomass, the biomass fraction having reduced toxicity compared to the bacterial biomass, wherein the steps of the first and second processes are carried out, and therefore all details of the individual steps described above for the first and second processes also pertain to the third process described herein.

[0055] The third process therefore comprises the following steps: 3a. Suspending a bacterial biomass in a solution comprising a chelating agent to provide a first cell suspension; 3b. Incubating the first cell suspension for a predetermined period of time; 3c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction; 3d.Ca 2+ , Mg 2+ , Cu 2+ , Fe 2+ , Co 2+ , Al 2+ adding at least one divalent cation to the first biomass fraction, such as 3e. Lysing at least a portion of the bacterial cells in the first biomass fraction; wherein steps 3d and 3e may be performed in either order to provide the third biomass fraction; 3f. Incubating the third biomass fraction for a predetermined period of time; The process whereby the third biomass fraction has reduced toxicity compared to the bacterial biomass.

[0056] Figure 3 shows the steps of the third process. The details of the individual steps of suspension, incubation, separation, and other steps in the third process are the same as those specified above for the first and second processes.

[0057] All three of the above processes result in reduced toxicity compared to the bacterial biomass. Reduced toxicity can refer to a reduction in the lipopolysaccharide (LPS) content or potency of the biomass. Reduced toxicity can be measured by conventional cytotoxicity tests (e.g., the Limulus Amebocyte Lysate (LAL) test) or by quantitative methods, such as quantification of intact LPS molecules or markers derived from LPS molecules by liquid or gas chromatography followed by mass spectrometry, flame ionization detection, thermal conductivity detection, or other suitable detection methods.

[0058] A semiquantitative method can be used to release fatty acids from LPS using a chemical process, separate them by chromatography, and quantify the major fatty acids as LPS markers. The fatty acids on LPS are different from those on the major lipid populations and can be distinguished from normal fatty acids. The detoxified product has a reduced / attenuated LPS content and can therefore pass cytotoxicity tests.

[0059] A further advantage is that the resulting product has reduced outer membrane components, resulting in a product with a high protein content.

[0060] Furthermore, following the process of the present invention, endotoxins are dephosphorylated during the process, thereby attenuating their deleterious effects, resulting in a non-toxic product or a product of reduced toxicity compared to the bacterial biomass.

[0061] Further provided is a bacterial biomass fraction having a lipopolysaccharide (LPS) content of less than 5% w / w, more preferably less than 2% w / w, even more preferably less than 1% w / w of the bacterial biomass fraction.

[0062] Such bacterial biomass fractions may pass cytotoxicity tests due to the reduced / attenuated LPS content present in the bacterial biomass fraction, reducing the chance of adverse effects occurring to humans during handling or storage of the product.

[0063] The animal feed or human food product may comprise or consist of a bacterial biomass fraction having a lipopolysaccharide (LPS) content of less than 10% w / w of the bacterial biomass fraction, such as less than 7% w / w, more preferably less than 5% w / w, more preferably less than 2% w / w, more preferably less than 1% w / w.

[0064] Such products can pass cytotoxicity tests and can be fed directly to animals or provided as human food products (e.g., raw materials or final products).

[0065] The present invention has been described with reference to a number of embodiments. Those skilled in the art can combine elements from different embodiments as needed. All documents cited herein are incorporated by reference. [Example]

[0066] In an exemplary embodiment, bacterial biomass (e.g., as obtained by the processes exemplified in WO2017 / 080987 and WO2022 / 008478) is subjected to a first centrifugation (using an SPX centrifuge, operating at 9,000 rpm, with a discharge time set to 120 seconds, feeding 500 L per hour, resulting in 50-60 L of sediment per hour and 440-450 L of supernatant per hour) to provide a first bacterial biomass fraction enriched in dry matter (first sediment, 10-15% dry matter) and a first supernatant lean in dry matter (1-2% dry matter). This fraction is then diluted 5-fold using water containing 5 mM tetrasodium EDTA. The resulting material was heated to 60 °C and immediately subjected to a second centrifugation (using an Alfa Laval CLARA centrifuge, operating at 9,000 rpm, with a discharge time of 120 seconds, feeding 300 L per hour, resulting in 30-40 L of precipitate per hour and 460-470 L of supernatant per hour), providing a second bacterial biomass fraction (second precipitate, 10-15% dry matter) and a second supernatant (1-2% dry matter). In the second precipitate, an LPS content of 65-70% of the first precipitate was observed by quantification of the LPS marker using chromatography.

Claims

1. 1. A process for providing a biomass fraction from a bacterial biomass that has reduced toxicity to said bacterial biomass, said process comprising: 1a. Suspending a bacterial biomass in a solution comprising a chelating agent to provide a first cell suspension; 1b. Incubating the first cell suspension for a predetermined period of time; 1c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction; A process whereby the first biomass fraction has reduced toxicity compared to said bacterial biomass.

2. 1. A process for providing a biomass fraction from a bacterial biomass that has reduced toxicity to said bacterial biomass, said process comprising: 2a. Providing a second biomass fraction by lysis of at least a portion of the bacterial cells in the bacterial biomass; 2b. Incubating the second biomass fraction for a predetermined period of time; whereby the second biomass fraction has reduced toxicity compared to said bacterial biomass.

3. 1. A process for providing a biomass fraction from a bacterial biomass that has reduced toxicity to said bacterial biomass, said process comprising: 3a. Suspending a bacterial biomass in a solution comprising a chelating agent to provide a first cell suspension; 3b. Incubating the first cell suspension for a predetermined period of time; 3c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction; 3d. Ca ２＋ , Mg ２＋ , Cu ２＋ , Fe ２＋ , Co ２＋ , Al ２＋ adding at least one divalent cation to the first biomass fraction, such as 3e. Lysing at least a portion of the bacterial cells in the first biomass fraction; wherein steps 3d and 3e may be performed in either order to provide the third biomass fraction; 3f. Incubating the third biomass fraction for a predetermined period of time; whereby the third biomass fraction has reduced toxicity compared to said bacterial biomass.

4. 4. The process of claim 1, further comprising subjecting the bacterial biomass to an initial separation step and removing a first liquid fraction prior to step 1a or 3a.

5. 4. The process of claim 1, further comprising one or more steps of washing and centrifuging the first biomass fraction after step 1c or step 3c and before any of the subsequent steps 3d-3f.

6. 6. The process according to any one of claims 3 to 5, wherein at least one divalent cation is added to the first biomass fraction in the form of an aqueous solution of said divalent cation.

7. 7. The process according to any one of claims 1 to 6, wherein the incubation of the first cell suspension in step 1b or step 3b is carried out under one or more of the following conditions: a pH between 5 and 11, preferably between 7 and 9, more preferably between 7.5 and 8.5; a temperature between 5 and 90°C, preferably between 20 and 90°C, more preferably between 30 and 90°C; a time period between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minute and 60 minutes, and more preferably between 1 minute and 30 minutes; 10 ８ From 10 １４ cells / mL, preferably between 10 １０ From 10 １２ The concentration of cells in the first cell suspension in cells / mL.

8. 8. The process according to any one of claims 3 to 7, wherein the incubation of the second biomass fraction in step 2b or the third biomass fraction in step 3f is carried out under one or more of the following conditions: a pH between 5 and 11, preferably between 7 and 9, more preferably between 7.5 and 8.5; a temperature between 5 and 90°C, preferably between 20 and 60°C, more preferably between 30 and 50°C; a time period between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minute and 60 minutes, and more preferably between 1 minute and 30 minutes; 10 ８ From 10 １４ cells / mL, preferably between 10 １０ From 10 １２ The concentration of cells in the first cell suspension in cells / mL.

9. 9. The process according to any of claims 2 to 8, wherein the lysis step (2a; 3e) comprises one or more steps selected from heating, cooling, freeze-thawing, sonication, mechanical treatment such as homogenization, chemical treatment, enzymatic treatment, pressure, and filtration, preferably heating.

10. 10. The process according to any one of claims 1, 3 to 9, wherein the chelating agent is a metal ion chelating agent, preferably a divalent metal ion chelating agent, preferably EDTA, ethylenediamine, glycine, citric acid, gluconic acid, tartaric acid, hexametaphosphate, pyrophosphate, tripolyphosphate, phytic acid, free histidine, L-glutamic acid, N,N-diacetic acid, aspartic acid, citrate, gluconate, or a salt thereof, in particular a sodium salt thereof, such as sodium citrate (TSA), sodium gluconate; preferably sodium EDTA, EDTA, sodium citrate (TSA), sodium gluconate, or a mixture thereof.

11. A bacterial biomass fraction having a lipopolysaccharide (LPS) content of less than 5% w / w of the bacterial biomass fraction, more preferably less than 2% w / w, even more preferably less than 1% w / w, even more preferably less than 0.5% w / w, even more preferably less than 0.2% w / w, even more preferably less than 0.1% w / w.

12. The process according to any one of claims 1 to 10 or the bacterial biomass according to claim 11, wherein the bacterial biomass is derived from the fermentation of at least one methanotrophic bacterium, preferably Methylococcus capsulatus.

13. 13. An animal feed comprising or consisting of the bacterial biomass fraction of claim 12.

14. A human food product comprising or consisting of the bacterial biomass fraction of claim 12.

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