Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/06—Lysis of microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
  • A23L29/065—Microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor

Definitions

• the present invention relates to processes 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 below 5% w/w of said biomass fraction.
• Methylococcus capsulatus is a non-commensal bacterium found ubiquitously in nature. It metabolizes methane, e.g ., from natural gas, into biomass, CO2 and water. Being rich in protein, M. capsulatus can be used as a protein supplement in animal feed and is also of interest for human consumption. The fermentation of this bacterium as a protein source for both animal and human consumption may contribute to satisfying the world's need for dietary protein in a way which is more environmentally friendly than conventional protein production industries.
• LPS Bacterial lipopolysaccharides
• endotoxins Bacterial lipopolysaccharides
• LPS naturally occurs in bacterial cells and is a structural component thereof; LPS consists of long chains of polysaccharides, which are covalently bound to lipids.
• the bacterial cell membrane constitutes a double lipid layer interspersed with proteins.
• the outer leaflet is LPS, which includes lipid A, a phosphorylated lipid .
• the toxicity of LPS is hypothesised to be mainly due to lipid A, while the polysaccharide part of LPS is generally considered less toxic.
• LPS acts as a pyrogenic compound, generating fever if directly injected into an animal, and 1 to 2 micrograms intravenously injected are lethal to both humans and animals.
• Wassenaar et al. Wangaar TM, Zimmermann K. Eur J Microbiol Immunol (Bp). 2018 Aug 21;8(3) :63-69. doi : 10.1556/1886.2018.00017
• LPS and its toxicity when ingested or injected by animals or humans. Removal of LPS from biomass is described in FI129784.
• LPS lipopolysaccharides
• the resulting product is less harmful, in that it comprises reduced bacterial cell outer membrane component concentrations.
• the present invention relates to a process for providing a biomass fraction having reduced toxicity relative to bacterial biomass, the process comprising the steps of: la. suspending a bacterial biomass in a solution comprising a chelating agent, to provide a first cell suspension, lb. incubating the first cell suspension for a predetermined time, and lc. 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 a reduced toxicity compared to the bacterial biomass.
• the present invention relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, the process comprising the steps of: 2a. lysis of at least a portion of the bacterial cells in the bacterial biomass, so as to provide a second biomass fraction, and 2b. incubating the second biomass fraction for a predetermined time, whereby the second biomass fraction has a reduced toxicity compared to the bacterial biomass.
• the present invention relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to bacterial biomass, combining the process steps of the first and second aspects.
• the process comprising 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 time, 3c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, followed by 3d.
• steps 3d. and 3e. may take place in any order, so as to provide a third biomass fraction, and 3f. incubating the third biomass fraction for a predetermined time, whereby the third biomass fraction has a reduced toxicity compared to the bacterial biomass.
• divalent cation such as Ca 2+ , Mg 2+ , Cu 2+ , Fe 2+ , Co 2+ , Al 2+
• the present invention relates to a bacterial biomass fraction having a lipopolysaccharide (LPS) content below 5% w/w, more preferably below 2% w/w, more preferably below 1% w/w of said biomass fraction.
• LPS lipopolysaccharide
• the present invention relates to an animal feed comprising or consisting of the bacterial biomass fraction according to the fourth aspect.
• the present invention relates to a human food comprising or consisting of the bacterial biomass fraction according to the fourth aspect.
• Fig. 1 shows an overview of the first process according to the invention.
• Fig. 2 shows an overview of the second process according to the invention.
• Fig. 3 shows an overview of the third process according to the invention, where the process steps 3a-3e are shown with the optional washing and centrifugation steps included.
• biomass refers to a proteinaceous product, which may be in the form of a protein extract and comprises cell wall materials of single celled microorganisms from pure or mixed cultures of algae, yeasts, fungi, or bacteria.
• biomass fraction refers to a fraction, i.e., a part of the biomass.
• single cell protein commonly refers to a proteinaceous product isolated from single celled microorganisms.
• the proteinaceous product may be in the form of a biomass or a protein extract and comprises cell wall materials of single celled microorganisms from pure or mixed cultures of algae, yeasts, fungi, or bacteria.
• the single cell protein is traditionally used as an ingredient or a substitute for protein-rich foods and is suitable for human consumption or as animal feeds.
• Utilizing microorganisms to obtain biomass for use in feed and food results in a product that has a higher proportion of nucleic acids than conventional foods.
• concentration of nucleic acids present in SCP varies depending on the specific microorganism employed, generally about 5 to 18 percent nucleic acids (dry weight) are present in SCP.
• DM refers to "Dry Matter”.
• dry matter and “ash” content is determined according to the A.O.A.C. method (reference A.O.A.C. Standard, 1945).
• dry weight in the context of the dry weight of an M. capsulatus biomass, should be taken to mean the weight of the biomass after all water has been removed from it. This should not be taken to mean that all water has been removed in all embodiments of an M. capsulatus biomass according to the present invention, since water is present in some embodiments. It should rather be understood as a measure that can be used to reproducibly calculate whether a certain biomass falls within the scope of the biomass according to the present invention.
• reduced toxicity refers to a reduced toxicity of the biomass fraction produced by microorganisms, such as pure or mixed cultures of algae, yeasts, fungi, or bacteria, thereby improving the safety of handling and storage of the biomass fractions. This may be e.g., reducing the LPS content in the biomass fraction, thus the reduced toxicity may refer to a reduced LPS content in the biomass fraction compared to an untreated bacterial biomass.
• solution comprising a chelating agent refers to a solution comprising a chelating agent molecule, i.e., a molecule which is able to bond metal ions.
• Chelation typically involves the formation of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single metal ion.
• the invention provides various processes for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass.
• the biomass material (which is typically an aqueous biomass material) is suitably obtained from the fermentation of at least one microorganism, preferably wherein at least one of the microorganisms is a bacterial cell, preferably a methanotroph, more preferably M. capsulatus.
• the microorganism In a fermentation step, the microorganism, or mixture of microorganisms, metabolizes methane into aqueous biomass material, CO2, and water.
• the fermentation occurs in fermentation tanks, and the process is described in detail in, e.g., WO 2017/080987 and WO 2022/008478 hereby incorporated by reference.
• the biomass material is a single-cell protein (SCP) product. It comprises mainly of protein (ca. 60%), and lesser amounts of RIMA and DNA.
• SCP single-cell protein
• the biomass material When isolated from the fermentation step, the biomass material is an aqueous suspension. In this aqueous suspension, the majority of the solid component is cellular material from the microorganism.
• Other components e.g., proteins, nucleic acids, polysaccharides, lipids, LPS or other small molecules
• At least one of the microorganisms used in the fermentation step is suitably a bacterial cell, preferably a Gram negative bacterial cell, preferably a methanotroph, more preferably M. capsulatus. Therefore, the biomass is suitably M. capsulatus biomass.
• Methods capsulatus can mean any strain of bacteria belonging to the M. capsulatus species.
• the strain may be either naturally occurring or developed in a laboratory, such as a genetically modified strain.
• naturally occurring means that the strain has not been genetically modified using genetic engineering techniques. However, it may contain natural modifications or alterations in its genetic material compared to a reference strain, such as alterations that occur randomly during replication.
• the strain is naturally occurring.
• the strain is M. capsulatus (Bath), more preferably the M. capsulatus (Bath) identified under NCIMB 11132. However, it may also be M. capsulatus (Texas) or M. capsulatus (Aberdeen) or a different M. capsulatus strain which is currently known or will be discovered or characterized in the future.
• the methanotrophic bacteria may be provided in a co-fermentation together with one or more heterotrophic bacteria.
• the following heterotrophic bacteria may be particularly useful to co-ferment with M. capsulatus,- Ralstonia sp. ; Bacillus brevis; Brevibacillus agri; Alcaligenes acidovorans; Aneurinibacillus danicus and Bacillus firmus.
• Suitable yeasts may be selected from species of Saccharomyces and/or Candida.
• the preferred heterotrophic bacteria are chosen from Alcaligenes acidovorans (NCIMB 13287), Aneurinibacillus danicus (NCIMB 13288) and Bacillus firmus (NCIMB 13289) and combinations thereof.
• the methanotrophic bacteria and/or the heterotrophic bacteria may be genetically modified.
• the carbon source is converted by the microorganism(s) to biomass material.
• the carbon source comprises methane, and is e.g., natural gas, syngas, or biogas.
• the carbon source is dissolved in the fermentation medium. Fermentation suitably takes place in a U-loop reactor, as described in WO 2010/069313, hereby incorporated by reference.
• a suitable fermentation medium is described in e.g., WO 2018/158322 hereby incorporated by reference.
• the fermentation step has a relatively low Dry Matter content, e.g., below 5%.
• the co-fermentation of M. capsulatus with one or more other organisms may result in a biomass product containing an M. capsulatus biomass as well as a biomass of the one or more other organisms.
• the 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 or two or all three of: A. acidovorans, A. danicus, and B. firmus; more preferably any one or two or all three of: A. acidovorans (NCIMB 13287), A. danicus (NCIMB 13288), and B. firmus (NCIMB 13289).
• one or more bacteria selected from : Ralstonia sp., B. brevis, B. agri, A. acidovorans, A. danicus, and B. firmus,- preferably any one or two or all three of: A. acidovorans, A. danicus, and B. firmus; more preferably any one or two or all three of: A. acidovorans (NCIMB 13287), A.
• the dry matter content of the biomass material from the fermentation process is between 1- 3%.
• the biomass material may be clarified, and the supernatant from the clarification step may be recycled to the fermenter. Pellets are thus obtained with a dry matter content of 10-18%.
• the fermentation broth in the fermenter may preferably continuously be provided with the required amounts of water and nutrient salts, such as ammonium/ammonia, magnesium, calcium, potassium, iron, copper, zinc, manganese, nickel, cobalt and molybdenum in the form of sulphates, chlorides or nitrates, phosphates and pH controlling components, i.e. acids and/or bases, as normally used by the skilled person, e.g.
• H2SO4 sulphuric acid
• HNO3 nitric acid
• NaOH sodium hydroxide
• KNO3 potassium nitrate
• the biomass material produced from fermentation of natural gas will typically comprise from 60 to 80% by weight crude protein; from 5 to 20% by weight crude fat; from 3 to 12% by weight ash; from 3 to 15% by weight nucleic acids (RNA and DNA).
• RNA and DNA nucleic acids
• the biomass material is subjected to a diafiltration step at this point, to lower the dry matter content to around 6%.
• the first process relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, said process comprising the steps of: la. suspending a bacterial biomass in a solution comprising a chelating agent, to provide a first cell suspension, lb. incubating the first cell suspension for a predetermined time, lc. 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 a reduced toxicity compared to the bacterial biomass.
• Fig. 1 shows the steps of the first process.
• the bacterial biomass is suspended in a solution comprising a chelating agent to provide a first cell suspension.
• the bacterial biomass is derived from fermentation of at least one methanotrophic bacteria, preferably M. capsuiatus.
• the process may further comprise the step of subjecting the bacterial biomass to an initial separation step to remove a first liquid fraction.
• the solution comprising a 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, Hexametaphosphoric acid, Pyrophosphoric acid, Tripolyphosphoric acid, Phytic acid, Free histidine, L-glutamic acid, N,N-diacetic acid, Aspartic acid, citrate, gluconate, or salts thereof, particularly sodium salts thereof, e.g. sodium citrate (TSA), sodium gluconate; preferably sodium EDTA, EDTA, sodium citrate (TSA), sodium gluconate, or mixtures thereof.
• TSA sodium citrate
• TSA sodium gluconate
• TSA sodium gluconate
• TSA sodium gluconate
• TSA sodium gluconate
• TSA sodium gluconate
• TSA
• the first cell suspension is incubated for a predetermined time.
• the incubation of the first cell suspension takes place 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 of between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minutes and 60 minutes, more preferably between 1 minute and 30 minutes, a concentration of the cells in the first cell suspension of between 10 8 and 10 14 cells/mL, preferably between 10 10 and 10 12 cells/mL.
• 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 a reduced toxicity compared to the bacterial biomass.
• One or more steps of washing and centrifugation of the first biomass fraction may occur after the third step.
• the combined chelation and heating (incubating) steps will maximise/promote the shedding of the outer membrane of the Gram-negative bacteria, therefore shedding of endotoxins while also reducing the nucleic acids in the bacterial biomass product.
• the second process relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, said process comprising the steps of:
• FIG. 2 shows the steps of the first process.
• lysis of at least a portion of the bacterial cells in the bacterial biomass is carried out, to provide a second biomass fraction.
• the lysis step comprises one or more steps selected from heating, cooling, freeze-thawing, sonication, mechanical treatment such as homogenisation, chemical treatment, enzymatic treatment, pressure and filtering, preferably heating.
• the parameters for homogenization are 800 bar and a flow of 80-100 litres/hour. This step is preferably followed by UHT treatment.
• the second biomass fraction is incubated for a predetermined time, whereby the second biomass fraction has a reduced toxicity compared to the bacterial biomass.
• the incubation of the second biomass fraction takes place 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 of between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minutes and 60 minutes, more preferably between 1 minute and 30 minutes, a concentration of the cells in the first cell suspension of between 10 8 and 10 14 cells/mL, preferably between 10 10 and 10 12 cells/mL.
• the third process relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, in which the steps of the first and second processes are carried out. Therefore, all details of the individual steps described above for the first and second processes are also relevant for the third process described herein.
• the third process thus comprises the steps of:
• steps 3d. and 3e. may take place in any order, to provide a third biomass fraction
• Fig. 3 shows the steps of the third process. 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.
• the reduced toxicity may refer to reducing the lipopolysaccharide (LPS) content or potency of the biomass.
• the reduced toxicity may be measured by a conventional cytotoxicity test (such as the limulus amoebocyte lysate (LAL) test), or a quantitative method such as quantification of the intact LPS molecule, or a marker derived from the LPS molecule, by e.g. liquid or gas chromatography followed by mass spectrometry, flame ionisation detection, thermal conductivity detection or another suitable detection method,
• a further advantage is that the resulting product will have reduced outer membrane components and leads to a product with higher protein content.
• the endotoxin is dephosphorylated during the process, thereby their negative effect is attenuated, resulting in a non-toxic product or in a product with reduced toxicity compared to the bacterial biomass.
• bacterial biomass fraction having a lipopolysaccharide (LPS) content below 5% w/w, more preferably below 2% w/w, more preferably below 1% w/w of said biomass fraction.
• LPS lipopolysaccharide
• Such a bacterial biomass fraction can pass the cytotoxicity test due to the reduced/attenuated LPS content present in the bacterial biomass fraction and reduces the chances of adverse effects occurring in the human body during handling or storage of the product.
• An animal feed or human food product may comprise or consist of the bacterial biomass fraction having a lipopolysaccharide (LPS) content below 10% w/w, such as below 7% w/w, more preferably below 5% w/w, more preferably below 2% w/w, more preferably below 1% w/w of said biomass fraction.
• LPS lipopolysaccharide
• Such a product is able to pass the cytotoxicity test. It can be directly fed to animals or provided as a food product (e.g. as ingredient or final product) for humans.
• a bacterial biomass (as obtained by the processes exemplified in e.g. WO 2017/080987 and WO 2022/008478) is subjected to a first centrifugation (using an SPX centrifuge running at 9,000 rpm and discharge time set to 120 seconds, and fed with 500 L per hour and delivering 50-60 L of precipitate per hour and 440-450 L of supernatant per hour) to provide a first bacterial biomass fraction enriched in dry matter (first precipitate, 10-15 % dry matter), and a first supernatant low in dry matter (1-2 % dry matter). This fraction is diluted by 5 fold using water containing 5 mM tetrasodium EDTA.
• the resulting material is heated to 60 °C and immediately subjected to a second centrifugation (using an Alfa Laval CLARA centrifuge running at 9,000 rpm and discharge time set to 120 seconds, and fed with 300 L per hour and delivering 30-40 L of precipitate per hour and 460-470 L of supernatant per hour) to provide a second bacterial biomass fraction (second precipitate, 10- 15 % dry matter) and a second supernatant (1-2 % dry matter).
• second precipitate a content of LPS amounting to 65-70 % relative to the first precipitate on a dry matter basis by quantification of an LPS marker was observed using chromatography.

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• 2023
  • 2023-11-06 EP EP23802182.8A patent/EP4615955A2/de active Pending
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