Nucleic acid product and process

Abstract

Methods of isolating nucleic acid enriched products from an aqueous biomass material are provided. After the heat shock treatment and incubation steps, the aqueous biomass material is separated to provide a first concentrated biomass material and a first liquid fraction. Various purification steps are performed to make full use of all products in the biomass.

Description

Technical Field

[0001] The present invention relates to a method for separating a nucleic acid-rich product from an aqueous biomass material. After a heat shock treatment and an incubation step, the aqueous biomass material is separated to provide a first concentrated biomass material and a first liquid fraction. Various purification steps (e.g., nanofiltration) may then be performed. Background Art

[0002] The methanotrophic bacterium Methylococcus capsulatus is a non-symbiotic bacterium that is ubiquitous in nature. It metabolizes methane (e.g., from natural gas) into biomass, CO 2 and water. Methylococcus capsulatus is rich in protein and 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 animal and human consumption can help meet the world's need for dietary protein in a more environmentally friendly manner than conventional protein production industries.

[0003] In order for an industrial production process to be both economically and environmentally viable, as many valuable products as possible should be separated, for example, from waste streams.

[0004] WO 2018 / 115042 relates to a method for removing nucleic acids from biomass. However, the method described therein does not provide clearly separated fractions, because complexes are formed in the disrupted biomass material in which protein and nucleic acid components are firmly bound.

[0005] The heat shock treatment described in L. Jørgensen et al. (J. Larsen & L. Jørgensen, Applied Microbiology and Biotechnology Vol. 45, pp. 137-140 (1996)) describes how to reduce the nucleic acid content in the protein product from a culture of Methylococcus capsulatus. However, the document does not describe any use or fractionation of the waste stream from this heat shock treatment.

[0006] Related publications include WO2021 / 071895 and WO2004 / 029076.

[0007] The aim is to increase the utilization of waste streams from biomass production in this way. Summary of the invention

[0008] Surprisingly it has been found that waste streams from biomass production contain useful levels of by-products, in particular nucleic acids, which can be easily separated from the process. It has also been found that by pre-treating the biomass, the biomass can be separated into a protein fraction and a nucleic acid fraction.

[0009] Therefore, in a first aspect, the present invention relates to a method for isolating a nucleic acid-enriched product from an aqueous biomass material, the method comprising the following steps:

[0010] subjecting the aqueous biomass material to a heat shock treatment to provide a heat shock treated biomass material;

[0011] incubating the heat-shocked biomass material at an elevated temperature for a predetermined period of time;

[0012] separating the aqueous biomass material in a first separation step to provide a first concentrated biomass material and a first liquid fraction;

[0013] then:

[0014] subjecting the first liquid fraction from the first separation process to a first nanofiltration (NF) step to provide a first NF permeate rich in minerals and a first NF retentate rich in nucleic acids and peptides having nucleic acids dissolved therein;

[0015] or

[0016] subjecting the first liquid fraction from the first separation step to a second filtration step to provide a second permeate enriched in minerals and a second retentate enriched in nucleic acids having nucleic acids dissolved therein;

[0017] then:

[0018] subjecting the nucleic acid-enriched retentate from the second filtration step to spray drying to obtain a nucleic acid-enriched product;

[0019] or

[0020] subjecting the nucleic acid-enriched second retentate from the second filtration step to a second nanofiltration (NF) step to obtain a nucleic acid- and peptide-enriched second NF-retentate and a mineral-enriched second NF-permeate;

[0021] or

[0022] The mineral-enriched second permeate from the second filtration step is subjected to a third nanofiltration step to obtain an RNA-enriched third-NF retentate and a mineral-enriched third NF-permeate.

[0023] This and other aspects of the invention are set forth in the following patent claims, the drawings and the examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1a An overview of the process of the present invention up to the first separation process point is shown.

[0025] Figure 1b Shows the Figure 1aOverview of one option (option 1b) of the process of the invention in which the supernatant / permeate (i.e. the liquid fraction) of the first separation process in a first nanofiltration treatment is subjected to a high concentration (upconcentrate) of the supernatant / permeate into a fraction enriched in nucleic acids (NA) and peptides (first NF retentate) and a first NF permeate fraction containing minerals and water.

[0026] Figure 1c Shows the Figure 1a An overview of the process of the present invention is performed on the precipitate / retentate of the first separation step in.

[0027] Figure 1d Shows the Figure 1a Summary of a second option (option 1d) of the process of the invention in which the supernatant / permeate of the first separation process is subjected to a second filtration step (MF / UF) to obtain a second permeate enriched in minerals and a second retentate enriched in nucleic acids in which the nucleic acids are dissolved. There are then 3 options:

[0028] Option 1d-1: MF / UF second retentate can be sent directly to the spray dryer

[0029] Option 1d-2: Sending the MF / UF second retentate to a second NF-separation step to provide a second NF-retentate enriched in nucleic acids and peptides and a second NF-permeate enriched in minerals.

[0030] Option 1d-3: Send the MF / UF second permeate to a third nanofiltration unit where the third NF-retentate is enriched in RNA and the third NF-permeate contains only minerals and water.

[0031] Detailed disclosure

[0032] Throughout the present specification, the abbreviation "DM" means "dry matter". In the present specification, the terms "dry matter" and "ash" content are determined according to the AOAC method (reference AOAC Standard, 1945).

[0033] As used herein, the term "dry weight", in the context of Methylococcus capsular biomass dry weight, is understood to mean the weight of the biomass after all moisture has been removed. This should not mean that all water has been removed in all embodiments of the Methylococcus capsular biomass according to the present invention, because water is present in some embodiments. Instead, it should be understood to be a measure that can be used to repeatedly calculate whether a certain biomass falls within the biomass scope according to the present invention.

[0034] The term "nucleic acid" as used herein includes DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) and fragments thereof. Nucleic acids are composed of monomers called nucleotides.

[0035] When a product such as a permeate or retentate is described as being "enriched" in a certain component after a given treatment step, it is to be understood that it is "enriched" in that component compared to the component prior to the treatment step. For example, when a first liquid fraction is subjected to a second filtration step to provide a second permeate enriched in minerals and a second retentate enriched in nucleic acids having nucleic acids dissolved therein, the mineral content of the second permeate is enriched (=increased) relative to the first liquid fraction.

[0036] "Enriched in nucleic acids" or "enriched in nucleic acids" means that during the relevant processing step the ratio of nucleic acids to minerals, proteins or dry matter has been increased, for example by at least 30%.

[0037] Microfiltration is filtration through membranes with a pore size > 0.1 μm and a molecular weight cut-off (MWCO) > 100 kDa.

[0038] Ultrafiltration is filtration through membranes with a pore size of 10 nm to 100 nm and a molecular weight cut-off (MWCO) of 1 kDa to 500 kDa.

[0039] Nanofiltration is membrane filtration with a pore size of 0.1 nm to 20 nm and a molecular weight cut-off (MWCO) of 0.01 kDa to 10 kDa.

[0040] As described above, the present invention provides methods for isolating nucleic acid-enriched products from aqueous biomass materials.

[0041] Fermentation

[0042] The aqueous biomass material is suitably obtained from the fermentation of at least one microorganism, preferably wherein the at least one microorganism is a bacterial cell, preferably a methanotrophic bacterium, more preferably Methylococcus capsulatus.

[0043] In the fermentation step, the microorganism or mixture of microorganisms metabolizes the methane into aqueous biomass material, CO 2 The fermentation is carried out in a fermenter and the process is described in detail in, for example, WO 2017 / 080987 and WO 2022 / 008478 (hereby incorporated by reference).

[0044] Biomass material is a single cell protein (SCP) product. It contains most of the protein (about 60%), and lesser amounts of RNA and DNA. When separated from the fermentation step, the biomass material is an aqueous suspension. In this aqueous suspension, most of the solid components are cellular material from microorganisms. Other components (e.g., proteins, nucleic acids, polysaccharides, lipids or other small molecules) can be dissolved or suspended in the aqueous phase.

[0045] The at least one microorganism used in the fermentation step is suitably a bacterial cell, preferably a methanotrophic bacterium, more preferably Methylococcus capsulatus. Thus, the biomass is suitably Methylococcus capsulatus biomass.

[0046] As used herein, the term "Methylococcus capsulatus" or "Methylococcus capsulatus" may refer to any bacterial strain belonging to the species Methylococcus capsulatus. 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, compared to a reference strain, it may contain natural modifications or changes in its genetic material, such as changes that occur randomly during the replication process. Preferably, the strain is naturally occurring. Also preferably, the strain is Methylococcus capsulatus (Bath), more preferably Methylococcus capsulatus (Bath) identified according to NCIMB 11132. However, it may also be Methylococcus capsulatus (Texas) or Methylococcus capsulatus (Aberdeen) or a different Methylococcus capsulatus strain currently known or to be discovered or characterized in the future.

[0047] Methanogens can be provided together with one or more heterotrophic bacteria in co-fermentation. The following heterotrophic bacteria may be particularly suitable for Methylcoccus capsulatus, Ralstonia sp, Bacillus brevis, Brevibacillus agri, Alcaligenes acidovorans, Aneurinibacillus danicus and Bacillus firmus. Suitable yeasts can be selected from species of Saccharomyces and / or Candida. Preferably, heterotrophic bacteria are selected from Alcaligenes acidovorans (NCIMB 13287), Aneurinibacillus danicus (NCIMB 13288) and Bacillus firmus (NCIMB 13289) and combinations thereof. Methanogens and / or heterotrophic bacteria can be genetically modified.

[0048] In the fermentation step, the carbon source is converted into a biomass material by one or more microorganisms. Suitably, the carbon source comprises methane, and is, for example, natural gas, synthesis gas or biogas. In the fermentation step, the carbon source is dissolved in a fermentation medium. As described in WO2010 / 069313 (incorporated hereby by reference), the fermentation is suitably carried out in a U-loop reactor. Suitable fermentation media are described in, for example, WO2018 / 158322 (incorporated hereby by reference). The fermentation step has a relatively low dry matter content, for example, less than 5%.

[0049] Co-fermentation of Methylococcus capsulatus with one or more other organisms can produce a biomass product comprising Methylococcus capsulatus biomass and biomass of the one or more other organisms.

[0050] In some embodiments, Methylococcus capsulatus is fermented in combination with one or more bacteria selected from the group consisting of Ralstonia species, Bacillus brevis, Bacillus brevis soil, Bacillus acidovorans, A. danicus, and Bacillus firmus; preferably any one or two or all three of Bacillus acidovorans, A. danicus, and Bacillus firmus; more preferably any one or two or all three of Bacillus acidovorans (NCIMB 13287), A. Danicus (NCIMB 13288), and Bacillus firmus (NCIMB13289).

[0051] Further details of the fermentation process are described in WO 2020 / 245197 and WO 2020 / 249670 (which are hereby incorporated by reference).

[0052] The dry matter content of the biomass material from the fermentation process is between 1% and 1.5%. After the biomass material is harvested from the fermentation process, the biomass material can be clarified, and the supernatant from the clarification step can be recycled to the fermentor. The precipitation thus obtained has a dry matter content of 10-18%. Preferably, the fermentation broth in the fermentor can be continuously provided with a desired amount of water and nutrients, such as ammonium / ammonia, magnesium, calcium, potassium, iron, copper, zinc, manganese, nickel, cobalt and molybdenum, phosphate and pH control components in the form of sulfate, chloride or nitrate, i.e., acids and / or alkalis commonly used by those skilled in the art, such as sulfuric acid (H 2 SO 4 ), nitric acid (HNO 3 ), sodium hydroxide (NaOH), potassium nitrate (KNO 3 ). The latter is also a suitable nitrogen source for Methylococcus capsulatus. Specific details of the fermentation process and substrates etc. are described in WO 2000 / 70014 and WO 2010 / 069313 (which are incorporated by reference).

[0053] The biomass material produced by natural gas fermentation will contain 60 to 80% by weight crude protein, 5 to 20% by weight crude fat, 3 to 12% by weight ash, 3 to 15% by weight nucleic acids (RNA and DNA).

[0054] Optionally, the biomass material is subjected to a dilution step at this point to reduce the dry matter content to about 6%.

[0055] Heat shock

[0056] In the first step, the aqueous biomass material is subjected to a heat shock treatment to provide a heat shock treated biomass material. The heat shock treatment is believed to activate endogenous nucleases, thereby reducing RNA and DNA.

[0057] Suitably, the heat shock treatment is carried out at a temperature of at least 70°C, preferably at least 80°C, more preferably at least 90°C. The heat shock treatment may be carried out for a period of between 1 second and 200 seconds, preferably between 5 seconds and 100 seconds, more preferably between 10 seconds and 50 seconds. In a preferred embodiment, the heat shock treatment is carried out at about 90°C for a period of about 10 seconds. The heat shock treatment may be carried out by passing the aqueous biomass material through a heated coil.

[0058] Incubation

[0059] The heat-shocked biomass material is then incubated at high temperature for a predetermined period of time. During the incubation process, larger RNA and DNA molecules are decomposed. The incubation of the heat-shocked biomass material is suitably carried out at a temperature of at least 40°C, preferably at least 50°C, and more preferably at least 60°C. The incubation of the heat-shocked biomass material can be carried out for a period of time between 1 minute and 200 minutes, preferably between 5 minutes and 100 minutes, and more preferably between 10 minutes and 50 minutes. In a preferred embodiment, the incubation is carried out at 50-60°C for a period of time of 20-30 minutes.

[0060] After incubation, the aqueous biomass material typically has a protein content of 50-80% w / w and a nucleic acid content of 4-12% w / w.

[0061] First separation step

[0062] After heat shock and incubation, the aqueous biomass material is subjected to a first separation process to provide a first concentrated biomass material and a first liquid fraction.

[0063] The first separation process can be an ultrafiltration (UF) process, a microfiltration (MF) process or a centrifugation process, preferably a UF process. If a MF process is used, it usually has a MCWO of 100-2000 kDa. If a UF process is used, it usually has a MWCO of 10-100 kDa.

[0064] If the first separation process is UF, the UF permeate is the first liquid fraction and the UF retentate is the first concentrated biomass material.

[0065] Prior to the first separation step, the aqueous biomass material may be washed and diluted.

[0066] If the first separation process is a centrifugation process, the first liquid fraction is the supernatant and the first concentrated biomass material is the precipitate.

[0067] The first liquid fraction comprises nucleic acids, peptides, vitamins, minerals and amino acids. The first separation process may have a molecular weight cutoff (MWCO) greater than 1,000,000 Daltons, such as greater than 1,200,000 Daltons, for example greater than 1,500,000 Daltons. At this point in the process, the dry matter content is typically 1-5%.

[0068] Advantageously, the pH of the first liquid fraction may be adjusted to a pH between 3 and 5, such as about pH 4, before being further processed.

[0069] The first concentrated biomass material typically has a protein content of 50-80% w / w and a nucleic acid content of 0-5% w / w.

[0070] refer to Figure 1b :The first liquid fraction from the first separation process is then subjected to a second nanofiltration (NF) step to provide a first NF permeate rich in minerals and a first NF retentate rich in nucleic acids and peptides having nucleic acids dissolved therein. The NF step suitably has a MWCO between 100 Da and 2000 Da.

[0071] The nucleic acid- and peptide-rich first NF retentate from the second NF step typically has a dry matter content of 3-15%, a protein content of 40-75% w / w and a nucleic acid content of 10-40% w / w.

[0072] refer to Figure 1d : The first liquid fraction is transferred from the first separation step to a second filtration step to provide a second permeate enriched in minerals and a second retentate enriched in nucleic acids having nucleic acids dissolved therein. The ratio of nucleic acids to minerals in the second retentate is increased by at least 30% compared to the same ratio in the first liquid fraction.

[0073] The second separation step may be an ultrafiltration (UF) step or a microfiltration (MF) step, and is preferably a UF step.

[0074] The UF membrane is a semi-permeable dynamic disc filter with a pore size of 20 nm or 5000 Da MWCO. The device also includes a backflush system that returns a portion of the permeate to the membrane every 20 seconds. In this system, the rotating disc limits membrane clogging and the formation of a polarization layer. The maximum time for performing UF is 24 hours, such as less than 24 hours, such as less than 15 hours, such as less than 11 hours, such as less than 8 hours, such as less than 6 hours, such as less than 4 hours.

[0075] Option 1d-1

[0076] In one option, the nucleic acid-enriched second retentate from the second filtration step is used directly for spray drying. This product is enriched in nucleic acids, peptides and minerals.

[0077] Option 1d-2

[0078] In another option, the second retentate enriched in (MF / UF) nucleic acids from the second filtration step is subjected to a second nanofiltration (NF) step to obtain a nucleic acid-enriched product (second NF-retentate).

[0079] The second nanofiltration (NF) stage also provides a second NF permeate that is rich in minerals. The second NF permeate typically contains monovalent ions.

[0080] The second NF-retentate enriched in nucleic acids comprises 0.5-10% (w / w) dry matter, preferably 0.5-5% (w / w) dry matter, more preferably 2-3% (w / w) dry matter. The product generally comprises 10-90% (w / w) nucleic acids, preferably 40-80% (w / w) nucleic acids, more preferably 50-70% (w / w) nucleic acids.

[0081] The method may further comprise the step of drying, preferably spray drying, the nucleic acid-enriched second NF-retentate to provide a dried nucleic acid-enriched product.

[0082] The nucleic acid-enriched product can be used directly or further processed as a food or feed ingredient according to pending regulations. In particular, the nucleic acid-enriched product can be used as an ingredient for baby food or infant formula.

[0083] Option 1d-3

[0084] In another option, the mineral-enriched MF / UF second permeate is subjected to a third nanofiltration (NF) step to obtain a third NF-retentate enriched in RNA and a third NF-permeate enriched in minerals. The third NF permeate typically comprises monovalent ions.

[0085] The ratio of RNA to mineral in the third NF-retentate was increased by at least 30% compared to the same ratio in the second permeate.

[0086] The RNA-enriched third NF-retentate comprises 0.5-20% (w / w) dry matter, preferably 0.5-10% (w / w) dry matter, more preferably 6-7% (w / w) dry matter. The product typically comprises 10-90% (w / w) RNA, preferably 40-80% (w / w) RNA, more preferably 50-70% (w / w) RNA.

[0087] The method may further comprise the step of drying, preferably spray drying, the RNA-enriched third NF-retentate to provide a dried RNA-enriched product.

[0088] The RNA-enriched product can be used directly or further processed according to pending regulations for use as an ingredient in food or feed. In particular, the RNA-enriched product can be used as an ingredient in baby food or infant formula. Example 1

[0089] Methylococcus capsulatus biomass material can be prepared by the method described in Larsen and Jørgensen, Appl. Microbiol. Biotechnol., 1996. Briefly, Methylococcus capsulatus, such as Methylococcus capsulatus Bath (NCIMB 11132), is grown in a bioreactor in a suitable medium with methane as a carbon source. The cells are then heat-shocked by heating the cell suspension to 90° C. for 10 seconds, followed by incubation of the cells at 60° C. for 20 minutes at pH 7.0.

[0090] References

[0091] J. Larsen & L. Jørgensen Applied Microbiology and Biotechnology volume 45, pages 137–140 (1996)

[0092] WO 2018 / 115042.

Claims

1. A method for isolating a nucleic acid-rich product from an aqueous biomass material, the method comprising the following steps: - subjecting the aqueous biomass material to a heat shock treatment to provide a heat shock treated biomass material; - incubating the heat-shocked biomass material at an elevated temperature for a predetermined period of time; - separating the aqueous biomass material in a first separation step to provide a first concentrated biomass material and a first liquid fraction; then: o subjecting the first liquid fraction from the first separation process to a first nanofiltration (NF) step to provide a first NF permeate enriched in minerals and a first NF retentate enriched in nucleic acids and peptides having nucleic acids dissolved therein; or o subjecting the first liquid fraction from the first separation process to a second filtration step to provide a second permeate enriched in minerals and a second retentate enriched in nucleic acids having nucleic acids dissolved therein; then: subjecting the nucleic acid-enriched second retentate from the second filtration step to spray drying to obtain a nucleic acid-enriched product; or subjecting the nucleic acid-enriched second retentate from the second filtration step to a second nanofiltration (NF) step to obtain a nucleic acid- and peptide-enriched second NF-retentate and a mineral-enriched second NF-permeate; or - Subjecting said mineral-enriched second permeate from said second filtration step to a third nanofiltration step to obtain a third NF-retentate enriched in RNA and a third NF-permeate enriched in minerals.

2. The method according to claim 1, further comprising the step of drying, preferably spray drying, the first NF-retentate, the second NF-retentate and / or the third NF-retentate to provide a dried nucleic acid-enriched product.

3. The method according to any one of the preceding claims, wherein the first separation process is an ultrafiltration (UF) process, a microfiltration (MF) process or a centrifugation process, preferably a UF process.

4. The method according to any one of the preceding claims, wherein the second separation process is an ultrafiltration (UF) process, a microfiltration process or a centrifugation process, and preferably a UF process.

5. The method according to any one of the preceding claims, wherein the aqueous biomass material is subjected to washing and dilution prior to the first separation step.

6. The method according to any one of the preceding claims, wherein the heat shock treatment is performed at a temperature of at least 70°C, preferably at least 80°C, more preferably at least 90°C.

7. The method according to any one of the preceding aspects, wherein the heat shock treatment is performed for a time between 1 second and 200 seconds, preferably between 5 seconds and 100 seconds, more preferably between 10 seconds and 50 seconds.

8. The method according to any one of the preceding claims, wherein the incubation of the heat-shocked biomass material is performed at a temperature of at least 40°C, preferably at least 50°C, more preferably at least 60°C.

9. The method according to any one of the preceding claims, wherein the incubation of the heat-shocked biomass material is carried out for a time between 1 minute and 200 minutes, preferably between 5 minutes and 100 minutes, more preferably between 10 minutes and 50 minutes.

10. The method according to any one of the preceding claims, wherein the aqueous biomass material is obtained from the fermentation of at least one microorganism, preferably wherein the at least one microorganism is a bacterial cell, preferably a methanotroph, more preferably Methylococcus capsulatus.

11. A process according to any one of the preceding claims, wherein the aqueous biomass material is obtained from the fermentation of a mixture of methanotrophic bacteria and one or more heterotrophic bacteria.

12. The process according to claim 11, wherein the heterotrophic bacteria are selected from the group consisting of Ralstonia sp., Bacillus brevis, Brevibacillusagri, Alcaligenes acidovorans, Aneurinibacillus danicus and Bacillus firmus.

13. The method according to any one of the preceding claims, wherein the nucleic acid-enriched product comprises 0.5-10% (w / w) dry matter, preferably 0.5-5% (w / w) dry matter, more preferably 0.5-2% (w / w) dry matter.

14. The method of claim 13, wherein the nucleic acid-enriched product typically comprises 10-90% (w / w) nucleic acids, preferably 40-80% (w / w) nucleic acids, more preferably 50-70% (w / w) nucleic acids.

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