Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/20—Proteins from microorganisms or unicellular algae
• • 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
  • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
• • 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
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/195—Proteins from microorganisms
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/14—Ultrafiltration; Microfiltration
  • B01D61/145—Ultrafiltration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/14—Ultrafiltration; Microfiltration
  • B01D61/147—Microfiltration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/14—Ultrafiltration; Microfiltration
  • B01D61/149—Multistep processes comprising different kinds of membrane processes selected from ultrafiltration or microfiltration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/58—Multistep processes
• • 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
  • C12N1/08—Reducing the nucleic acid content
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D2311/00—Details relating to membrane separation process operations and control
  • B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D2311/00—Details relating to membrane separation process operations and control
  • B01D2311/26—Further operations combined with membrane separation processes
  • B01D2311/2688—Biological processes

Definitions

• the present invention relates to a method for providing one or more isolates from a biomass.
• the present invention relates to an industrial method for removing nucleic acids from a biomass, e.g. for producing single cell protein from cultivated methanotrophic bacteria with a reduced nucleic acid content.
• SCP single cell protein
• 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.
• the amount of nucleic acids present in SCP varies depending on the specific microorganism employed, generally about 5 to about 18 percent nucleic acids (dry weight) are present in SCP.
• RNA, DNA and nucleic acid as such are not desired in the protein product, such as in the SCP product, as these compounds may have direct or indirect effect on the health of a mammal, such as humans or animals, e.g. by causing gout or gouty arthritis or kidney stones in the mammal .
• dietary RNA and DNA are decomposed into nucleic acid fragments in the intestinal lumen, and further decomposed into nucleotides and/or nucleosides and free purine and pyrimidine bases by nucleotide and/or nucleoside phosphatase enzymes in the mucosa.
• uric acid The metabolism of purine bases results in high levels of uric acid. Humans do not possess the enzyme uricase, which oxidizes uric acid to allantoin, a soluble and excretable metabolite. Consumption of a protein source high in nucleic acids results in hyperuricemia which is defined by abnormally high level of uric acid found in the blood. Uric acid has low solubility at physiological pH values thus forming crystals of uric acid that can be retained in the joints and kidneys, causing gout or gouty arthritis and kidney stones.
• nucleic acids may in excessive or uncontrolled amounts be considered as biogenic substances and are regarded as a limiting factor in the use of SCP derived from algae, yeasts, fungi and bacteria in food products for human nutrition.
• the normal plasma uric acid concentration in men is 5.1 ⁇ 0.9 mg ml 1 and in women is approximately 1 mg ml 1 less.
• the Recommended Daily Allowance for protein is 65 grams per day for a 70-kilogram adult male and the Protein Calorie Advisory Group of the United Nations System recommends that the amount of nucleic acid ingested per day from microbial protein should be less than 2 grams with the total nucleic acid from all sources not exceeding a total of 4 grams per day.
• processes that have been used in the past for nucleic acid removal such as enzymatic treatment, acid treatment, base treatment and heat shock, affect the SCP product in terms of flavour, odour and colour and since the content of nucleic acid, fragments thereof and nucleotides and/or nucleosides is very high in the traditional SCP product the SCP product, becomes unattractive for food, as it requires to have a mild flavour, odour and colour so as not to influence the palatability of the food or feed.
• an object of the present invention relates to a simplified method for providing one or more isolates, in particular, a SCP product where the nucleic acids (e.g. DNA and/or RNA) are removed without affecting the SCP product.
• nucleic acids e.g. DNA and/or RNA
• one aspect of the invention relates to a method for providing one or more fraction(s) from a biomass material, the method comprises the steps of: (i) providing the biomass material;
• an aspect of the present invention relates to a method for providing a Single Cell Protein product (SCP product) from a biomass material wherein said SCP product comprising a reduced amount of nucleic acids relative to the naturally occurring amount of nucleic acid in the biomass material, the method comprising the steps of: (i) providing the biomass material;
• step (v) optionally, combining the first retentate obtained in step (iii) with the vitamins, minerals and/or amino acids obtained in step (iv), providing the SCP product comprising a reduced amount of nucleic acids relative to the naturally occurring amount of nucleic acids.
• Another aspect of the present invention relates to a method for removing nucleic acids from a biomass material, the method comprises the steps of: (i) providing the biomass material;
• step (v) optionally, combining the first retentate obtained in step (iii) with the vitamins, minerals and/or amino acids obtained in step (iv), providing a fraction wherein the nucleic acids have been removed.
• the second retentate obtained from the ultrafiltration comprises the DNA and RNA
• the second permeate obtained from the ultrafiltration comprises the vitamins, minerals, and amino acids (AA).
• the first retentate and the second permeate are then combined providing the SCP product according to the present invention.
• Figure 2 shows an embodiment of the present invention for providing various fractions from downstream processing of fermented bacterial single cell protein resulting in fractions comprising cell debris, suspended solids, fat, proteins and/or peptides,
• the fermented single cell protein may preferably be bacterial single cell protein which, in figure 2, may be obtained from the fermentation of methanotrophic bacteria in a reactor (1), such as a U-Loop reactor (1).
• the U-Loop reactor (1) may, during the fermentation, be supplied with methane (1), e.g. provided in the form of biogas, mineral solutions (3), and the needed oxygen (4).
• methane (1) e.g. provided in the form of biogas, mineral solutions (3), and the needed oxygen (4).
• excess CO2 produced by the methanotrophic bacteria may be discharged from the reactor (1) through the outlet (5).
• the biomass may be harvested and transferred to the homogenizer (6) which disrupts the cells liberating intracellular proteins and/or peptides, minerals, salts, vitamins etc.
• the disrupted biomass is transferred to a decanter (7) where the cell debris fraction (8) may be taken out.
• the biomass may subsequently be transferred to a clarifier (9) for removing suspended solids (10), like suspended cell debris.
• the clarified biomass may subsequently be subjected to a fat separator (11) providing a fat fraction (12).
• the biomass may then be subjected to first separation process (13) comprising either membrane filtration, e.g. by microfiltration (13), or chromatographic separation, e.g. by affinity chromatography (13), providing a protein and/or peptide fraction (14) - a first fraction.
• SCP Single cell protein
• Microbial protein such as single cell protein (SCP) requires cultivation of microorganisms in a fermentation tank.
• SCP single cell protein
• the biomass material according to the present invention may be provided by the fermentation process described in WO 2010/069313; WO 2000/70014; or US 2004/0241790, preferably the biomass material is provided by the fermentation process as described in WO 2010/069313, which are all incorporated by reference.
• the biomass material may be subjected to further downstream processing.
• the first fraction obtained in step (iii) may be combined with the fourth fraction obtained in step (iv), providing a fifth fraction.
• a biomass material is provided with reduced amount of nucleic acid.
• the viscosity of the disrupted biomass material may be too high complicating pumping and processing of the disrupted biomass material, particularly in the first separation process and/or the second separation process.
• the viscosity of the disrupted biomass material may be reduced.
• the decanting process and/or the clarifying process may mainly remove cells and cell debris from the biomass material, providing a disrupted biomass material with reduced cell debris.
• the disrupted biomass material obtained from the decanting process (the supernatant obtained from decanting) may constitute a disrupted biomass material with reduced content of cells and cell debris.
• a disrupted biomass material with reduced content of cells and cell debris comprise less than 10% (w/w) cell or cell debris, such as below 8%, e.g. below 7%, such as below 6%, e.g. below 5%, such as below 4%, e.g. below 3%, such as between 1.5-10%, e.g. between 2-9%, such as between 2.5-8%, e.g. between 3-7%, such as between 3.5-6%, e.g. between 4-5%.
• the fat fraction obtained from the fat removal process mainly consists of fatty acids that may be used for the production of soaps, cosmetics, and industrial mold release agents.
• the fat fraction may also find use in foodstuffs because they are inexpensive and may add texture and "mouth feel" to processed foods (convenience food).
• the first separation process comprises a first membrane filtration or a first chromatographic separation process.
• the chromatographic separation process may include a column chromatographic separation process.
• the column chromatographic separation process includes a Packed Bed Chromatography, stirred tank adsorption, Fluidized Bed Chromatography and/or Expanded Bed Chromatography.
• the column chromatographic separation process may be Expanded Bed Chromatography.
• the chromatographic separation process may include affinity
• the chromatographic separation process may be affinity chromatography or mixed mode chromatography.
• Another preferred embodiment of the present invention relates to a method for providing a SCP product from a biomass material wherein said SCP product comprising a reduced amount of nucleic acids relative to the naturally occurring amount of nucleic acids in the biomass material, the method comprising the steps of: (i) providing the biomass material;
• step (v) optionally, combining the first retentate obtained in step (iii) with the vitamins, minerals and/or amino acids obtained in step (iv), providing the SCP product comprising a reduced amount of nucleic acids relative to the naturally occurring amount of nucleic acids.
• the biomass material provided in step (i) may be provided from a fermentation tank, preferably from a U-Loop fermenter (preferably as described in WO 2010/069313).
• the methods according to the present invention are shown to reduce the content of nucleic acids in the fermentation product by at least 10%, relative to the naturally occurring amount of nucleic acid in the biomass material ; such as at least 20%, e.g. at least 30%, such as at least 40%, e.g. at least 50%, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 90%, such as at least 95%, e.g. at least 98%.
• a further preferred embodiment of the present invention relates to a method for removing nucleic acids from a biomass material, the method comprises the steps of:
• step (v) optionally, combining the first retentate obtained in step (iii) with the vitamins, minerals and/or amino acids obtained in step (iv), providing a SCP product wherein 10 the nucleic acids have been removed.
• nucleic acids relates to biopolymers, or large biomolecules, essential for all known forms of life. Nucleic acids, include DNA
• Nucleic acids are 15 made from monomers known as nucleotides. In the present context, the terms
• nucleotide and “nucleoside” may be used interchangeably and the simple difference is that nucleosides can be considered nucleotides without a phosphate group.
• removing nucleic acids relates to removal of at least 20 10% of the nucleic acids naturally present in the biomass material (resulting in the
• Suitable biocatalysts used in the process and the fermenter according to the invention may preferably be living cells, e.g. microorganisms of natural origin, i.e. wild types, specially selected mutated types or genetically modified types that may be used to produce single cell protein, enriched single cell protein, proteins or peptide extracts, cell extracts, or preparations containing particular beneficial substances to be used for example for food or
• the biocatalysts are preferably living microorganisms. Fermentation of the microorganisms may be carried out using pure cultures or using blends or a mixture of different microorganisms, e.g. for production of baker's yeast, single cell protein (SCP). The fermentation process may also result in biotransformations (i.e. microbial conversion of different chemicals to other useful chemicals), or production of intracellular or extracellular enzymes, proteins or hormones for use in different industries or in certain products, (e.g. pharmaceuticals, nutraceuticals or compounds for use as diagnostic or analytic agents).
• the preferred bacteria for use in the invention are those capable of producing single cell protein, especially a culture comprising methanotrophic bacteria.
• the biomass material may be a single-cell protein material.
• the single-cell protein material, and the biomass material comprises a methanotrophic bacteria.
• the methanotrophic bacteria may optionally be combined with one or more species of other bacteria, e.g. heterotrophic bacteria.
• the fermenter may be used for the fermentation of methylotrophic fungi or yeasts such as Pic ia stipitis or Pichia pasto s.
• P. stipitis and P. pastoris are both capable of metabolizing methanol and may be suitable for potential GMO-production.
• the preferred methanotrophic bacteria are species of the Methylococcaceae family, especially Methyiococcus capsulatus, which utilize methane or methanol as a carbon source and e.g. ammonia, nitrate or molecular nitrogen as a nitrogen source for protein synthesis.
• the methanotrophic bacteria may be selected from the family Methylococcaceae or the family Methylocystaceae.
• the biomass material comprises a Methyiococcus strain.
• the Methyiococcus strain is Methyiococcus capsulatus.
• capsulatus Therefore, it may be useful to co-ferment one or more strains of heterotrophic bacteria with the methanotrophic bacteria for digesting higher hydrocarbons (alcohols, carboxylic acids, etc.) e.g. ethanol, acetate, citrate, etc. or degradation products of partially digested dead or decaying biomass.
• hydrocarbons e.g. ethanol, acetate, citrate, etc.
• degradation products of partially digested dead or decaying biomass e.g. ethanol, acetate, citrate, etc.
• capsulatus a co-fermentation of M. capsulatus with Alcaligenes acidovorans (NCIMB 13287),
• NCIMB 13288 Aneurinibacillus danicus
• NCIMB 13289 Bacillus firmus
• the process of cell disruption involves 5 homogenization of the biomass material, subjecting the biomass material to ball milling or shear forces.
• the cell disruption involves homogenization and the
• homogenization may be a high-pressure homogenization.
• the size exclusion used during ultrafiltration will generally be in the range of about 100,000 Daltons.
• biomass material may be cooled, preferably to a
• Homogenization may be carried out in a conventional high-pressure homogenizer in which the cells may be disrupted by first pressurizing, and then depressurizing the inside of the homogenizer.
• Homogenization may preferably be high-pressure homogenization which involves a change in pressure of the biomass material .
• change in pressure of the biomass material may be a pressure drop in the range of from 200 to 2,500 bar, such as in the range of 400 to 2,000 bar, e.g. in the range of 600 to 1,500 bar, such as in the range of 1,000 to 1,300 35 bar, e.g. in the range of 1,200 to 1,250 bar, such as in the range of 1,300 to 2,200 bar, e.g. in the range of 1400 to 2,000 bar, such as above 1,200 bar, e.g. above 1,250 bar, such as above 1,500 bar, e.g. about 2,000 bar.
• the process of cell disruption provided in step (ii) may be performed under controlled temperature conditions, preferably at a
• a single step of drop-in pressure may be preferred, however, in an embodiment of the present invention the drop-in pressure may be stepped, such as comprising two or more steps. If two or more steps are provided the drop-in pressure may start with the highest pressure drop and followed by a decrease in successive drops in pressure according to the number of steps.
• the homogenization process herein described results in a disrupted biomass material comprising disrupted cells.
• the disrupted cells may be present in an amount of at least 80% by weight (20% of the cells remain undisrupted), preferably at least 90% by weight, even more preferably at least 95% by weight, even more preferred at least 98% by weight.
• the disrupted biomass material may be a relatively viscous protein slurry containing soluble and particulate cellular components, such as proteins; cell debris; RNA; DNA; vitamins; minerals: and amino acids (such as free amino acids).
• the first separation process may have a molecular weight cut-off value (MWCO) of greater than 1,000,000 Dalton, such as greater than 1,200,000 Dalton, e.g. greater than 1,500,000 Dalton.
• MWCO molecular weight cut-off value
• the first membrane filtration may involve a ceramic membrane filter material.
• the first membrane filtration may be a dynamic disc filter.
• the disrupted biomass material may be supplied to a first separation process which involves a microfiltration process, preferably using ceramic membrane filter material. From the microfiltration process a first retentate may be provided, said first retentate comprising proteins and/or cell debris. From the microfiltration process a first permeate may be provided, said first permeate comprising RNA, DNA, vitamins, minerals and amino acids (free amino acids).
• the first permeate obtained from the first separation process may be subjected to a second treatment separating the nucleic acids from the vitamins, minerals, and the amino acids (free amino acids).
• the second membrane filtration may be an ultrafiltration providing a second retentate comprising the nucleic acids and a second permeate comprising vitamins, minerals and/or amino acids (free amino acids).
• the second membrane filtration may have a molecular weight cut-off value (MWCO) in the range of 10,000-100,000 Dalton, such as in the range of 25,000-75,000 Dalton. Furthermore, it is preferred that the second membrane filtration may have a pore size in the range of 0.002-0.1 ⁇ , such as a pore size in the range of 0.005-0.05 ⁇ , e.g. a pore size in the range of 0.0075-0.01.
• MWCO molecular weight cut-off value
• the second membrane filtration may involve an organic polymer membrane, such as polysulfones, poly (styrenes), PVDF (polyvinylidene fluoride) and PAN (polyacrylonitrile) including styrene-containing copolymers such as acrylonitrile-styrene, butadiene-styrene and styrene-vinylbenzylhalide copolymers, polycarbonates, cellulosic polymers,
• organic polymer membrane such as polysulfones, poly (styrenes), PVDF (polyvinylidene fluoride) and PAN (polyacrylonitrile) including styrene-containing copolymers such as acrylonitrile-styrene, butadiene-styrene and styrene-vinylbenzylhalide copolymers, polycarbonates, cellulosic polymers,
• the second membrane filtration may involve an organic polymer membrane.
• the ceramic membrane used in the first membrane filtration (and/or in the second membrane filtration) may be based on alumina, titanium, zirconia oxides, silicon carbide or some glassy materials.
• the second treatment may involve precipitation of the nucleic acids.
• the nucleic acids may be precipitated from the vitamins, minerals, and amino acids by the addition of an organic alcohol, preferably an alcohol selected from ethanol or isopropanol.
• the organic alcohol such as isopropanol or ethanol, may be removed from the supernatant by evaporation or distillation.
• the first permeate may be supplied to a second separation process which preferably involves an ultrafiltration process, preferably using ceramic membrane filter material; or a precipitation process where the nucleic acids are precipitated using ethanol or isopropanol.
• a second retentate may be provided, said second retentate comprising nucleic acids (RNA and DNA).
• a second permeate may be provided, said second permeate comprising vitamins, minerals and amino acids (free amino acids).
• the ceramic membrane used in the first separation step; in the second separation step or in both separation steps may be placed under pressure to improve capacity and/or effectivity of the membrane.
• a turbulent flow may be imparted to the biomass material in contact with the membrane and this turbulent flow agitates the liquids adjacent to the membrane and permits a higher content of solids in the retentate.
• the one or more fraction or the SCP product may be pasteurized.
• the separation processes e.g. the membrane filtrations (microfiltration and
• the method may further comprise the step
• step (vii) inactivation of the enzyme added in step (vi).
• the process of cell disruption in step (ii); the first separation process in step (iii); the second treatment in step (iv); the preparation of the SCP product in step (v); the enzyme treatment in step (vi) and/or the enzyme inactivation in step (vii) is/are performed under controlled temperature conditions, preferably at a temperature of less than 50°C, particularly preferably from 25 to 50°C, e.g. from 25 to 35°C.
• the method according to the present invention may furthermore, comprise the step of adding one or more unsaturated fatty acids, such as ARA, DHA and/or EPA, to the one or more fraction obtained from the present invention, such as the first retentate obtained in step (iii), to the second permeate obtained in step (iv) and/or to the SCP product obtained in step (v).
• one or more fraction, the first retentate, the second permeate, and/or the SCP product may be combined with one or more unsaturated fatty acids, such as ARA, DHA and/or EPA, preferably the SCP product may be combined with DHA.
• a preferred embodiment of the present invention relates to one or more fraction or a SCP product comprising a biomass material and a reduced content of nucleic acids, relative to the naturally occurring amount of nucleic acids in the biomass material.
• enzymatic degradation of the nucleic acids is not used on too large amount of nucleic acids, since the process simply results in the degradation of the nucleic acids to nucleotides and nucleosides but does not remove the components and the challenge with joints and kidneys, gout or gouty arthritis and kidney stones may still occur.
• the one or more fraction or the SCP product may comprise less than 90 mg nucleic acids per gram biomass material on a dry-matter basis, such as less than 75 mg/g biomass material, e.g. less than 50 mg/g biomass material, such as less than 25 mg/g biomass material, e.g. less than 1 mg/g biomass material, such as less than 750 ⁇ g/g biomass material, e.g. less than 500 ⁇ g/g biomass material, such as less than 100 ⁇ g/g biomass material, e.g. less than 10 ⁇ g/g biomass material.
• the one or more fraction or SCP product may comprise a single-cell protein material.
• the one or more fraction or the SCP product comprises a methanotrophic bacteria.
• the protein content of the SCP product of the present invention may be higher than prior art products where the nucleic acids are simply kept in the SCP product or where only enzymatic degradation of the nucleic acids has been introduced.
• the one or more fraction or the SCP product according to the present invention may comprise at least 50% protein on a dry-matter basis, such as at least 60% protein on a dry-matter basis, e.g. at least 70% protein on a dry-matter basis, such as at least 80% protein on a dry-matter basis, e.g. at least 90% protein on a dry-matter basis, such as at least 95% protein on a dry-matter basis, e.g. in the range of 50-95% protein on a dry- matter basis, such as in the range of 60-85% protein on a dry-matter basis, e.g. in the range of 65-75% protein on a dry-matter basis, such as in the range of 68-83% protein on a dry-matter basis.
• a dry-matter basis such as at least 60% protein on a dry-matter basis, e.g. at least 70% protein on a dry-matter basis, such as at least 80% protein on a dry-matter basis,
• the one or more fraction or SCP product may be supplemented with one or more fatty acids.
• the one or more fraction or SCP product may comprise one or more unsaturated fatty acids, such as ARA, DHA and/or EPA, preferably, the one or more fraction or SCP product may comprise DHA.
• the content of unsaturated fatty acids in the one or more fraction or SCP product may be dependent on the intended use of the product.
• the one or more fraction or SCP product comprises 0.5-15% (w/w) on a dry-matter basis of the
• the one or more fraction or SCP product according to the present invention may be used directly in, or as an ingredient for, food or feed products, the one or more fraction or SCP product may usually be further processed e.g. to remove excess water from the product. During the further processing the one or more fraction or SCP product may also be subjected to an additional drying step to provide a dry product comprising one or more fraction or SCP.
• the dry one or more fraction or SCP product may have a moisture content of 15% or less, such as 10% or less, e.g. 8% or less, such as 5% or less.
• the additional drying step may be provided by using a spray dryer.
• the one or more fraction or SCP product according to the present invention may be used directly as a food or a feed product; or it may be used as an ingredient for a food or a feed product.
• the feed may be a fish feed or animal feed or human feed, preferably a fish feed or an animal feed.
• the one or more fraction or the SCP product may be used for food or feed or to be delivered in order to improve or optimize the health, performance or well-being of humans or animals, such as, but not limited to cloven hoofed animals (e.g. cattle, goats, sheep, pigs, etc.), poultry (e.g. fowls, chicken, ducks, goose/geese, turkey, etc.), fish (e.g. salmon, halibut, trout, cod, or other species bred in captivity) or shellfish (e.g. molluscs such as mussels, oysters, shrimps, prawns, lobsters, or scallops).
• cloven hoofed animals e.g. cattle, goats, sheep, pigs, etc.
• poultry e.g. fowls, chicken, ducks, goose/geese, turkey, etc.
• fish e.g. salmon, halibut, trout, cod, or other species bred in captivity
• the biomass material may preferably be derived from a single cell protein material, particularly comprising methanotrophic bacteria.
• the preferred strain of methanotrophic bacteria being Methylococcus capsulatus (NCIMB 11132) is provided from NCIMB (National Collection of Industrial, Food and Marine Bacteria, Aberdeen, Scotland).
• NCIMB National Collection of Industrial, Food and Marine Bacteria, Aberdeen, Scotland.
• M. capsulatus can only oxidize hydrocarbons into the corresponding alcohols, aldehydes and carboxylic acids, but cannot oxidize higher hydrocarbons completely to carbon dioxide and water or utilize them for biomass production three other strains Alcaligenes acidovorans (NCIMB 13287), Bacillus firmus (NCIMB 13289) and Aneurinibacillus danicus (NCIMB 13288) are also provided and used together with M. capsulatus.
• the carbon source preferred by the present invention may be natural gas, biogas, syngas, methane or methanol.
• the biomass material is then homogenised, and this process involves a change in pressure provided as a pressure drop 600-1500 bar leading to a disrupted biomass material.
• This process of homogenisation is not illustrated in figure 1, but is performed after harvesting of the biomass material and before applying the biomass material to the first separation process, the membrane filtration (MF).
• the provided disrupted biomass material is subjected to MF using a semi-permeable dynamic disc filter with a pore size of 0.5 ⁇ with 3 discoidal ceramic membranes.
• This device includes a backflush system that sends a part of the permeate back to the membrane every 20 sec. In this system, the rotating discs limit the membrane clogging and the formation of a polarisation layer.
• the MF is performed on a maximal period of 24 hours e.g. for a period less than 24 hours, e.g. for a period less than 15 hours, e.g. for a period less than 11 hours, e.g. for a period less than 8 hours, e.g. for a period less than 6 hours, e.g. for a period less than 4 hours.
• the resulting first retentate obtained from the MF process comprises proteins and cell debris and the first permeate comprises the DNA, RNA and vitamins, minerals and amino acids.
• the first permeate is then applied to a second membrane filtration being an ultrafiltration (UF).
• the UF membrane is a semi-permeable dynamic disc filter with a pore size of 20nm or 5000 Da MWCO.
• This device also includes a backflush system that sends a part of the permeate back to the membrane every 20 sec. In this system, the rotating discs limit the membrane clogging and the formation of a polarisation layer.
• the UF is performed on a maximal period of 24 hours e.g. for a period less than 24 hours, e.g. for a period less than 15 hours, e.g. for a period less than 11 hours, e.g. for a period less than 8 hours, e.g. for a period less than 6 hours, e.g. for a period less than 4 hours.
• the resulting second retentate obtained from the UF process comprises DNA and RNA, whereas the second permeate comprises the vitamins, minerals and amino acids.
• the liquid component of the biomass material passes through the semi-permeable ceramic MF membrane, hereafter mentioned as first permeate.
• the component that does not pass through the semi-permeable MF ceramic membrane, hereafter mentioned as the first retentate has a higher concentration of cell debris and proteins, than the first permeate.
• the MF first retentate is collected, and the first permeate is continued for further separation by contacting the first permeate with the semi-permeable UF ceramic membrane under the foregoing conditions until the desired second retentate composition is obtained.
• the second permeate is the filtration liquid that passes through the semipermeable UF ceramic membrane, hereafter mentioned as the second permeate.
• the component that does not pass through the semi-permeable UF ceramic membrane, hereafter mentioned as the second retentate has a higher concentration of nucleic acids, than the second permeate, the first permeate and the first retentate.
• the one or more fraction or SCP product may be provided by combining the retentate of the MF (the first retentate) with the permeate of the UF (the second permeate). This combination results in a fermentation product having a reduced nucleic acid content relative to the biomass material and to the corresponding products described in the prior art.
• the combined biomass fraction or SCP product comprising the first retentate and a second permeate of two consecutive filtrations, provides a biomass fraction or a SCP product enriched in cell wall debris, proteins, minerals, vitamins and amino acids, obtained from processing the fermentation biomass, preferably, comprising methanotrophic bacteria.
• dry matter and ash content was determined according to the A.O.A.C. method (reference A.O.A.C. Standard, 1945).
• a phenol-chloroform extraction is a liquid-liquid extraction.
• a liquid-liquid extraction is a method that separates mixtures of molecules based on the differential solubility of the individual molecules in two different immiscible liquids. Liquid-liquid extractions are widely used to isolate DNA and total RNA (Agency for Toxic Substances and Disease Registry. Toxicological Profile for Chloroform. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service; 1997).
• organic polymer membranes with the appropriate adaptations, in either the first separation process and/or in the second separation process, can be used as well.
• Preferred organic polymers may be polysulfones, poly (styrenes), PVDF (polyvinylidene fluoride) and PAN (polyacrylonitrile) including styrene-containing copolymers such as acrylonitrile-styrene, butadiene-styrene and styrene-vinylbenzylhalide copolymers, polycarbonates, cellulosic polymers, polypropylene, poly (vinyl chloride), poly (ethylene terephthalate).
• the liquid is an aqueous phase.
• the aqueous phase may preferably be water.

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