Oxidation-stable biomass materials and methods

Abstract

A method of producing an antioxidant stable biomass material is provided. In particular, the method comprises the steps of: treating a concentrated biomass material with an antioxidant; and adjusting the pH of the concentrated biomass material to 7.0 or less. Through these steps, synergistic stability of the biomass material can be seen. Also provided is an oxidation-stable biomass material, as well as an aquatic feed product comprising the oxidation-stable biomass material.

Description

Technical Field

[0001] A method for producing an antioxidant-stabilized biomass material is provided. Specifically, the method comprises the following steps: treating a concentrated biomass material with an antioxidant; and adjusting the pH of the concentrated biomass material to 7.0 or below. Through these steps, a synergistic stabilization of the biomass material can be seen. The present invention also provides an oxidatively stabilized biomass material, and an aquatic feed product comprising the oxidatively stabilized biomass material. 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, carbon dioxide 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 both animal and human consumption may help meet the world's demand for dietary protein to some extent, and is more environmentally friendly than traditional protein production industries.

[0003] Typically, biomass materials include fats and transition metals. The presence of transition metals in biomass materials is a result of the fermentation medium used when producing the biomass material. Attempts have been made to chelate transition metals, such as by adding one or more chelating agents, but this has not been shown to reduce oxidation.

[0004] Both fats and transition metals contribute to the production of oxidation products, which in turn can reduce the quality of the product and its shelf life. In addition, biomass products are often subjected to thermal treatments during production (e.g., spray drying and granulation), which is also believed to initiate or accelerate oxidation. Other factors believed to accelerate oxidation include the accessibility of oxygen and light, and the moisture content of the biomass product.

[0005] Fish feed presents a particular problem since fish will not eat oxidized feed.It is an object of the present invention to provide methods and biomass products having improved palatability. Summary of the invention

[0006] The present inventors have found that by adding antioxidants and changing or controlling pH, biomass products can be protected from oxidation. The pH adjuster and antioxidant act synergistically to provide an improvement in stability that is not apparent when these ingredients are used alone. In other words, treatment with an antioxidant alone, an acidity regulator alone, or an acidity regulator to an incorrect pH will not show an improvement in oxidative stability.

[0007] Therefore, in a first aspect, the present invention relates to a method for producing an antioxidant stabilized biomass material, the method comprising the following steps:

[0008] a. fermenting at least one methanotrophic bacterium in a fermentation medium in the presence of a carbon source to provide a biomass material;

[0009] b. separating the biomass material in a first separation step to provide a concentrated biomass material and a first liquid fraction;

[0010] c. performing inactivation treatment on the concentrated biomass material;

[0011] d. Optionally, homogenizing the concentrated biomass material;

[0012] Wherein step c. and step d. can be performed in any order,

[0013] The method comprises the following additional steps:

[0014] e. treating the concentrated biomass material with an antioxidant after step b; and

[0015] f. After step b, adjusting the pH of the concentrated biomass material to pH 7.0 or below.

[0016] Also provided is an oxidatively stable biomass material in the form of a dry powder or granules, comprising, by dry weight:

[0017] - 60-75% protein; preferably 65-72% protein;

[0018] - 1-10% fatty acids, preferably 7-9% fatty acids;

[0019] - 0.01-2% antioxidants;

[0020] - wherein when the biomass material is dissolved in water at a concentration of 100 to 400 g / L, the pH of the resulting solution is below 7.0.

[0021] Furthermore, an oxidatively stabilized biomass material, wherein the oxidatively stabilized biomass material has an oxidative stability that is at least two times, preferably at least three times, greater than the oxidative stability of the same biomass material that has not been stabilized; wherein the stabilization comprises the following steps:

[0022] - treating the biomass material with an antioxidant; and

[0023] - Adjust the pH of the biomass material to 7.0 or below.

[0024] The stabilization treatment can be performed in existing downstream production, but can also be implemented in other process lines for single-cell proteins from fermentations cultivated with methanotrophic bacteria.

[0025] This and other advantages of the invention are shown in the following patent claims, the drawings and the exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Various routes to the production of biomass according to the invention are described.

[0027] Detailed disclosure

[0028] In this document, the abbreviation "DM" means "dry matter". Specific implementation plan

[0029] Thus, a method for producing a biomass material that is stabilized against oxidation is provided. The oxidation stability tester (e.g. ML Oxipres from Mikrolab Aarhus A / S) is used to determine the oxidation stability of the biomass material. TM ) to determine the improved antioxidant stability.

[0030] The first step of the method is to (a.) ferment at least one methanotrophic bacterium in the presence of a carbon source in a fermentation medium to provide a biomass material.

[0031] The biomass material is a single cell protein (SCP) product. It contains a majority of proteins (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 cell materials from methanotrophic bacteria. Other components (e.g., proteins, nucleic acids, polysaccharides, lipids or other small molecules) may be dissolved or suspended in the aqueous phase.

[0032] At least one of the microorganisms used in the fermentation step is a methanotrophic bacterium, more preferably Methylococcus capsulatus. Thus, the biomass is suitably Methylococcus capsulatus biomass.

[0033] As used herein, the terms "Methylococcus capsulatus" or "Methylococcus capsulatus" may refer to any bacterial strain belonging to the species Methylococcus capsulatus. The strain may be naturally occurring or cultivated 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, its genetic material may contain natural modifications or changes, such as changes that occur randomly during replication. Preferably, the strain is naturally occurring. Also preferably, the strain is Methylococcus capsulatus (Bath), more preferably Methylococcus capsulatus (Bath) identified under NCIMB 11132. However, it may also be Methylococcus capsulatus (Texas) or Methylococcus capsulatus (Aberdeen) or a different strain of Methylococcus capsulatus currently known or to be discovered or characterized in the future.

[0034] Methane oxidizing bacteria can be provided in co-fermentation with one or more heterotrophs. The following heterotrophs can be particularly useful for co-fermentation with M. 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. Preferred heterotrophs are selected from Aneurinibacillus acidovorans (NCIMB 13287), Aneurinibacillus danicus (NCIMB 13288) and Bacillus firmus (NCIMB 13289) and combinations thereof. Methane oxidizing bacteria and / or heterotrophs can be genetically modified. In the co-fermentation, M. capsulatus accounted for 90-98%.

[0035] In the fermentation step, the carbon source is converted into a biomass material by a microorganism. 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. Suitably, the fermentation is carried out in a U-loop reactor, as described in WO 2010 / 069313, which is incorporated herein by reference. Suitable fermentation media are described in, for example, WO 2018 / 158322, which is incorporated herein by reference. The fermentation step has a relatively low dry matter content, for example, less than 5%.

[0036] Further details of the fermentation process are described in WO 2020 / 245197 and WO 2020 / 249670, which are incorporated herein by reference.

[0037] The second step in the method is (b) separating the biomass material in a first separation step to provide a concentrated biomass material and a first liquid fraction. The first separation step suitably comprises or consists of a centrifugation step, a membrane filtration step or a combination thereof, preferably, wherein the first separation step comprises or consists of a centrifugation step. The biomass is concentrated to provide a concentrated biomass material having a dry matter (DM) content of 5-25%, preferably 10-20%.

[0038] When the first separation step is a centrifugation step, it provides a first liquid fraction in the form of a supernatant.When the first separation step is a membrane filtration step, it provides a first fraction in the form of a filtration permeate.

[0039] The method further comprises step (c) of inactivating the concentrated biomass material. This is because all living host organisms need to be killed before obtaining the final product. The inactivation treatment may include one or more of ultra-high temperature (UHT) treatment, ultraviolet radiation treatment or sterile filtration treatment, and preferably ultra-high temperature (UHT) treatment. Preferably, the UHT treatment is carried out at a temperature of at least 120° C., preferably, 120 to 135° C. Suitably, the UHT treatment is carried out for 5 to 60 minutes.

[0040] In order to improve the uniformity of the concentrated biomass material, the method may include an optional step (d) of homogenizing the concentrated biomass material. Homogenization is typically performed in a homogenizer vessel at a pressure of 5 to 900 bar. During homogenization, large particles in the concentrated biomass material are broken down to obtain a more uniform distribution.

[0041] The inactivation and homogenization steps (steps c. and d.) can be performed in any order. Preferably, step c. is performed before step d.

[0042] At this point in the process, the pH of the concentrated biomass material is typically about pH 6.8-7.0.

[0043] According to the invention, the method comprises the following additional steps:

[0044] e. treating the concentrated biomass material with at least one antioxidant after step b; and

[0045] f. After step b, adjusting the pH of the concentrated biomass material to pH 7.0 or below.

[0046] As mentioned above, both steps are performed after step b., the separation step. Treatment with an antioxidant and pH adjustment work in a synergistic manner to improve the antioxidant properties of the biomass material. In other words, it is not enough to add an antioxidant or a pH adjuster alone.

[0047] In one aspect, steps e. and f. are performed simultaneously. In another aspect, step f. is performed before step e. Steps e. and f. may be performed after step d., or steps e. and f. may be performed before step d. In general, preferred methods include steps af. in sequence.

[0048] The following steps can be performed in the same container:

[0049] e. treating the concentrated biomass material with at least one antioxidant after step b; and

[0050] f. after step b, adjusting the pH of the concentrated biomass material to pH 7.0 or below,

[0051] Stirring is performed appropriately for at least 30 minutes. The temperature of the biomass material in the container may be obtained from the previous step (eg 20-40°C) or the biomass material in the container may be actively cooled to 7-10°C.

[0052] In a further aspect, both step c. and step d. are performed, and step c. is performed before step d.

[0053] The method may further comprise a water evaporation step between step b. and step c., between step b. and step d. and / or between steps c. and d. The evaporation step typically results in a DM between 20-40%, preferably 25-35%.

[0054] Step f. is suitably carried out by adding an acid or buffer to the concentrated biomass material to reach the desired pH. The measurement of the pH of the biomass material is within the purview of a person skilled in the art. Preferably, in step f., the pH of the biomass material is adjusted to pH 6.5 or below, such as 6.0 or below, 5.8 or below, preferably 5.5 or below. pH adjustment can be carried out by adding an inorganic acid or an organic acid, preferably an organic acid. In one aspect, pH adjustment is carried out by adding a C1-C5 organic acid, such as citric acid or propionic acid, preferably citric acid. Citric acid is preferred because it has polyacid functionality and is able to produce a stable buffer solution. Suitably, in step f., the pH of the biomass material is adjusted to pH 4.0 or above, preferably 4.5 or above.

[0055] The present invention can use a series of antioxidants, such as ascorbic acid, ascorbyl stearate, tocopherol, rosemary extract, propyl gallate, quinones such as tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), β-carotene, β-apo-8'-carotenal, carotenic acid, ethyl ester, β-apo-8'-, citric acid, isopropyl citrate, thiodipropionic acid, dilauryl thiodipropionate and stearyl citrate. Mixtures of two or more such antioxidants can also be used. Preferred antioxidants are ascorbic acid, ascorbyl stearate, tocopherol, rosemary extract, propyl gallate, quinones such as tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), β-carotene, β-apo-8'-carotenal, carotenic acid, ethyl ester, β-apo-8'-, thiodipropionic acid and dilauryl thiodipropionate. Tocopherol-containing antioxidants are most preferred, as are antioxidants of natural origin.

[0056] In one aspect, the method further comprises a final step g. drying the biomass material to provide a powdered biomass material. Suitably, the drying step is spray drying. The method may further comprise a step h. granulating the powdered biomass material to provide a granulated biomass material.

[0057] As described above, the method of the present invention provides an oxidatively stable biomass material. Preferably, the oxidatively stable biomass material has an anti-oxidative stability that is at least two times, preferably at least three times, greater than the anti-oxidative stability of the same biomass material that has not been subjected to steps e. and f.

[0058] The oxidatively stable biomass material in the form of dry powder or granules can be produced by the method of the present invention. The material comprises, by dry weight:

[0059] - 60-75%, preferably 65-72% protein;

[0060] - 1-10%, preferably 7-9% fatty acids;

[0061] - 0.01-2% antioxidants;

[0062] - wherein when the biomass material is dissolved in water at a concentration of 100 to 400 g / L, the pH of the resulting solution is below 7.0.

[0063] Also provided is an oxidatively stable biomass material, wherein the oxidatively stable biomass material has an oxidative stability that is at least two times, preferably at least three times, greater than the oxidative stability of the same biomass material that has not been stabilized; wherein the stabilization comprises the following steps:

[0064] - treating the biomass material with an antioxidant; and

[0065] - Adjust the pH of the biomass material to 7.0 or below.

[0066] The method and oxidatively stable biomass material may be used in an aquatic environment (ie as fish feed) where oxidation issues are of high importance. Accordingly, the present invention provides an aquafeed product comprising an oxidatively stable biomass material as described herein, preferably 10-30% DM.

[0067] Detailed Description

[0068] Methylococcus capsulatus biomass can be obtained by Larsen and Appl. Microbiol. Biotechnol., 1996. Methylococcus capsulatus, such as Methylococcus capsulatus Bath (NCIMB 11132), is grown in a bioreactor in a suitable medium using methane as a carbon source.

[0069] Figure 1 A downstream production process of biomass derived from fermentation based on the culture of a methanotrophic strain is shown. The downstream production concentrates the biomass from 1-2% DM to about 10-20% DM (see Figure 1 Steps 1 to 3). The product is then heat treated at 120-135°C (step 4) and then homogenized at 6-900 bar (step 5). The product can then, if desired, be spray dried (step 7) and granulated (step 8).

[0070] The present invention implements additional processing steps, see Figure 1 , where the concentrated biomass (step 3) is treated with an antioxidant and the pH is changed / controlled somewhere in the existing process. There are 5 possible production pathways for the antioxidant and pH treatment. All 5 possibilities are in Figure 1 The most common downstream process is from step 1 to step 8, where processing can be carried out in a storage tank in step 6 before spray drying.

[0071] The pH is usually adjusted before adding the antioxidant. The pH can be adjusted with an acid, preferably citric acid, but propionic acid can be used as an alternative. The pH is adjusted to below 7.0. Preferably below 6.0. Adding antioxidant: Weigh the antioxidant in an amount of 0.05-3 g / l and add after the pH is adjusted. It is usually necessary to stir the container for treatment. Cooling is optional.

[0072] The present invention provides single cell proteins (SCP) derived from fermentation with cultured methanotrophic bacteria that are resistant to oxidation when used in, for example, fish feed formulations. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Five production pathways useful in the present invention are described.

[0075] The first process line includes:

[0076] 1. Fermenter with 1-2% DM

[0077] 2. Separator to concentrate biomass to 10-20% DM

[0078] 3. Concentrated biomass

[0079] 4a. UHT treatment at 120-135°C

[0080] 5a. Homogenization of biomass at 5-900 bar

[0081] 6a. Balance tank, where pH adjuster and antioxidant are added to treat the biomass (pH is adjusted to below 7.0 and antioxidant is added in the range of 0.05-3g / l)

[0082] 7. Spray drying biomass - producing powder products P1

[0083] 8. Make powder into granules - produce granular products P2

[0084] The second process line includes:

[0085] 1. Fermenter with 1-2% DM

[0086] 2. Separator to concentrate biomass to 10-20% DM

[0087] 3. Concentrated biomass

[0088] 4. UHT treatment at 120-135°C

[0089] 5. Balance tank, where pH adjuster and antioxidant are added to treat the biomass (pH is adjusted to below 7.0 and antioxidant is added in the range of 0.05-3g / l)

[0090] 6. Homogenization of biomass at 5-900 bar

[0091] 7. Spray drying biomass - producing powder products P1

[0092] 8. Make powder into granules - produce granular products P2

[0093] The third process line includes:

[0094] 1. Fermenter with 1-2% DM

[0095] 2. Separator to concentrate biomass to 10-20% DM

[0096] 3. Concentrated biomass

[0097] 4b. UHT treatment at 120-135°C

[0098] 5b. Evaporate the biomass to a concentration of 25-35% DM

[0099] 6b. Homogenization of biomass at 5-900 bar

[0100] 6c. Balance tank, where pH adjuster and antioxidant are added to treat the biomass (pH is adjusted to below 7.0 and antioxidant is added in the range of 0.05-3g / l)

[0101] 7. Spray drying biomass - producing powder products P1

[0102] 8. Make powder into granules - produce granular products P2

[0103] The fourth process line includes:

[0104] 1. Fermenter with 1-2% DM

[0105] 2. Separator to concentrate biomass to 10-20% DM

[0106] 3. Concentrated biomass

[0107] 4c. Homogenization of biomass at 5-900 bar

[0108] 5c. UHT treatment at 120-135°C

[0109] 6c. Balance tank, where pH adjuster and antioxidant are added to treat the biomass (pH is adjusted to below 7.0 and antioxidant is added in the range of 0.05-3g / l)

[0110] 7. Spray drying biomass - producing powder products P1

[0111] 8. Make powder into granules - produce granular products P2

[0112] The fifth process line includes:

[0113] 1. Fermenter with 1-2% DM

[0114] 2. Separator to concentrate biomass to 10-20% DM

[0115] 3. Concentrated biomass

[0116] 4d. Homogenization of biomass at 5-900 bar

[0117] 5d. Balance tank, where pH adjuster and antioxidant are added to treat the biomass (pH is adjusted to below 7.0 and antioxidant is added in the range of 0.05-3g / l)

[0118] 6d. Inactivation of biomass by, for example, UV radiation or filtration

[0119] 7. Spray drying biomass - producing powder products P1

[0120] 8. Make powder into granules - produce granular products P2

[0121] Using ML Oxipres TM Exemplary Procedure for Determining Oxidative Stability.

[0122] program

[0123] 1. Select a suitable oxidation temperature (e.g. 80°C). Turn on the heater 20 minutes before the start of the experiment.

[0124] 2. Weigh an appropriate amount of sample into each glass container. (An amount containing 3-5 grams of fat usually gives reasonable results). Place a glass lid on top of the container.

[0125] 3. Place the glass container into a pressure vessel (high-pressure gas container).

[0126] 4. Make sure the O-ring and groove are clean. Install the top of the pressure vessel and hand-tighten the cap.

[0127] 5. Connect the pressure vessel to the filling station.

[0128] 6.a. Close the valve of the filling station.

[0129] b. Close the regulator outlet valve.

[0130] c. Open the master cylinder valve (slowly).

[0131] d. Adjust the outlet pressure of the regulator to 5 bar (70 psig).

[0132] e. Open the pressure vessel valve.

[0133] 7.a. Open the regulator outlet valve.

[0134] b. Turn the filling station valve to the FILL position

[0135] c. To flush out the air in the pressure vessel, turn the valve to the VENT position.

[0136] d. To repeat flushing, repeat steps b and c. (flushing 3 times will reduce the nitrogen content to less than 1%).

[0137] 8. Now fill the pressure vessel with oxygen (FILL). Use the cylinder pressure controller to reach 5 bar (70 psig) or less. Read the pressure of the pressure vessel on the display of the control unit.

[0138] 9. Close the filling station valve (OFF), and then close the pressure vessel valve.

[0139] 10. Observe the pressure for a few minutes to check whether the pressure vessel is properly tightened.

[0140] 11. If tightened, turn the filling station valve to (VENT) and disconnect the filling pipe from the pressure vessel.

[0141] 12. Place the pressure vessel into the block heater and start the test.

[0142] 13. Remember to close the cylinder main valve.

[0143] 14. After testing, open the valve. To reduce possible odors, ventilate through an odor trap (charcoal filter) or a fume hood.

[0144] Results Evaluation

[0145] Due to the consumption of oxygen, the pressure in the high-pressure gas container decreases. (Initially, the pressure increases due to heating). The signal from the pressure sensor is shown on the display. The signal is recorded as a function of time.

[0146] Characterize the induction period of the sample. The induction period (IP, in hours) can be found by plotting the signal as a function of time, plotting the tangent lines T1 and T2 of the curve. The time from START to the intersection is the IP. START is the time the pressure vessel is placed in the block heater. The induction period will be the time that elapses between placing the pressure vessel in the block heater and the breakpoint at a given temperature (e.g. 80°C).

[0147] Experimental results:

[0148] Initially, the focus was on peroxide (PV) and thiobarbituric acid (TBA) as oxidation parameters. Later, due to improved reliability, Oxipres, oxidative stability, was used by default.

[0149] The first test (Sample A, without pH adjuster or antioxidant) showed a reference stability of 17.5 hours (i.e. low stability). Adding antioxidant alone did not significantly improve stability (Sample B).

[0150] When the product was treated with a different combination of pH adjuster and antioxidant (samples CF), stability of up to 75 hours was observed. The procedure was tested on a larger scale (sample E) and stability of 61.3 hours was observed. The product was non-homogeneous, which shows that the treatment can be carried out in different types of biomass products and maintain oxidative stability. In sample F, another antioxidant from Vitablend was successfully tested, so the treatment is not dependent on one brand. Samples that absorb oxygen quickly have lower antioxidant properties (corresponding to the short time in the Oxipres test).

[0151] Table 1 - Oxidative stability of various biomass materials after various treatments. Oxipres values ​​(in hours) correspond to the induction period determined in the Oxipres method above.

[0152] Sample name Antioxidant treatment pH Adjusters pH Oxipres (hours) A(ref) none none 6.8 17.15 B(ref) Naturox IP2 none 7.5 19.5 C Naturox IP2 Citric Acid 5 75.75 D Naturox IP2 Citric Acid 4.6 80.2 E Naturox IP2 Citric Acid 5 61.3 F Vitablend Citric Acid 5 70.6 G(ref) none Citric Acid 7.5 18.75 H(ref) none Citric Acid 5.07 19.6 I(ref) none Sulfuric acid and citric acid 5.02 17.9 K none Propionic acid and citric acid 5.07 14.9 L(ref) none none 6.9 16.23 M Vitablend Citric Acid 5.3 69.4 N(ref) Vitablend none 7.1 18.53 O(ref) none Citric Acid 4.8 19.55

[0153] Provides the following numbered lists:

[0154] Aspect 1. A method for producing an antioxidant-stable biomass material, the method comprising the following steps:

[0155] a. fermenting at least one methanotrophic bacterium in a fermentation medium in the presence of a carbon source to provide a biomass material;

[0156] b. separating the biomass material in a first separation step to provide a concentrated biomass material and a first liquid fraction;

[0157] c. performing inactivation treatment on the concentrated biomass material;

[0158] d. Optionally, homogenizing the concentrated biomass material;

[0159] Wherein steps c. and d. can be performed in any order,

[0160] The method comprises the following additional steps:

[0161] e. treating the concentrated biomass material with at least one antioxidant after step b; and

[0162] f. After step b, adjusting the pH of the concentrated biomass material to pH 7.0 or below.

[0163] Aspect 2. The method according to aspect 1, wherein steps e. and f. are performed simultaneously.

[0164] Aspect 3. The method according to aspect 1, wherein step f. is performed after step e.

[0165] Aspect 4. The method according to any of the preceding aspects, wherein steps e. and f. are performed after step d, or wherein steps e. and f. are performed before step d.

[0166] Aspect 5. The method according to any of the preceding aspects, wherein the first separation step comprises a centrifugation step, a membrane filtration step or a combination thereof, preferably, wherein the first separation step comprises or consists of a centrifugation step.

[0167] Aspect 6. The method according to any of the preceding aspects, wherein the inactivation treatment is an ultra high temperature (UHT) treatment, preferably carried out at a temperature of at least 120°C.

[0168] Aspect 7. The method according to any of the preceding aspects, wherein step c. is performed before step d.

[0169] Aspect 8. The method according to any one of the preceding aspects, wherein, in step f., the pH of the biomass material is adjusted to pH 6.5 or below, such as 6.0 or below, 5.8 or below, preferably 5.5 or below.

[0170] Aspect 9. The method according to any one of the preceding aspects, wherein in step f., the pH of the biomass material is adjusted to pH 4.0 or above, preferably 4.5 or above.

[0171] Aspect 10. The method according to any one of the preceding aspects, wherein the pH adjustment is performed by adding a C1-C5 organic acid, such as citric acid or propionic acid, preferably citric acid.

[0172] Aspect 11. A method according to any of the preceding aspects, wherein the antioxidant is selected from ascorbic acid, ascorbyl stearate, tocopherol, rosemary extract, propyl gallate, quinones such as tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), β-carotene, β-apo-8'-carotenal, carotenic acid, ethyl ester, β-apo-8'-, citric acid, isopropyl citrate, thiodipropionic acid, dilauryl thiodipropionate, stearyl citrate, and a mixture of two or more such antioxidants.

[0173] Aspect 12. The method according to any of the preceding aspects, wherein the method further comprises a step of evaporating water between step b. and step c., between step b. and step d., and / or between step c. and d.

[0174] Aspect 13. The method according to any one of the preceding aspects, further comprising a final step g. drying the biomass material to provide a powdered biomass material.

[0175] Aspect 14. The method according to aspect 13, further comprising the step h. granulating the powdered biomass material to provide granulated biomass material.

[0176] Aspect 15. The method according to any of the preceding aspects, wherein at least one methanotrophic bacterium is Methylococcus capsulatus.

[0177] Aspect 16. The method according to any of the preceding aspects, wherein the biomass is Methylococcus capsulatus biomass.

[0178] Aspect 17. The method according to any one of the preceding aspects, wherein the fermentation step (a) comprises fermenting a mixture of methanotrophic bacteria and one or more heterotrophic bacteria.

[0179] Aspect 18. The method according to aspect 17, wherein the heterotrophic bacteria is selected from the group consisting of Ralstonia sp., Bacillus brevis, Brevibacillusagri, Alcaligenes acidovorans, Aneurinibacillusdanicus and Bacillus firmus.

[0180] Aspect 19. The method according to any of the preceding aspects, wherein the carbon source comprises methane, and is, for example, natural gas or biogas.

[0181] Aspect 20. The method according to any of the preceding aspects, wherein steps e. and f. provide an oxidatively stable biomass material whose anti-oxidative stability is at least twice, preferably at least three times, the anti-oxidative stability of the same biomass material not subjected to steps e. and f.

[0182] Aspect 21. The method according to any one of the preceding aspects, further comprising the step of recycling the first liquid fraction from the separation step (b) to the fermentation step (a).

[0183] Aspect 22. An oxidatively stable biomass material obtained from the fermentation of at least one methanotrophic bacterium, in the form of a dry powder or granules, comprising, by dry weight:

[0184] - 60-75%, preferably 65-72% protein;

[0185] - 1-10%, preferably 7-9% fatty acids;

[0186] - 0.01-2% antioxidants;

[0187] - wherein when the biomass material is dissolved in water at a concentration of 100 to 400 g / L, the pH of the resulting solution is below 7.0.

[0188] Aspect 23. An oxidatively stabilized biomass material obtained from fermentation of at least one methanotrophic bacterium, wherein the oxidatively stabilized biomass material has an antioxidant stability that is at least two times, preferably at least three times, greater than the antioxidant stability of the same biomass material that has not been stabilized; wherein the stabilization comprises the following steps:

[0189] - treating the biomass material with an antioxidant; and

[0190] - Adjust the pH of the biomass material to 7.0 or below.

[0191] Aspect 24. An aquaculture feed product comprising the oxidatively stable biomass material of any one of aspects 22-23, preferably 10-30% DM.

[0192] References

[0193] J.Larsen&L. Applied Microbiology and Biotechnology, Vol. 45, pp. 137-140 (1996).

Claims

1. A method for producing an antioxidant-stable biomass material, the method comprising the following steps: a. fermenting at least one methanotrophic bacterium in a fermentation medium in the presence of a carbon source to provide a biomass material; b. separating the biomass material in a first separation step to provide a concentrated biomass material and a first liquid fraction; c. performing inactivation treatment on the concentrated biomass material; d. Optionally, homogenizing the concentrated biomass material; Wherein steps c. and d. can be performed in any order, The method comprises the following additional steps: e. treating the concentrated biomass material with at least one antioxidant after step b; and f. After step b, adjusting the pH of the concentrated biomass material to pH 7.0 or below.

2. The method according to claim 1, wherein - Steps e. and f. are performed simultaneously, - Step f. is performed after step e., - step c. is performed before step d., - steps e. and f. are performed after step d., or - wherein steps e. and f. are performed before step d.

3. The method according to any one of the preceding claims, wherein the first separation step comprises a centrifugation step, a membrane filtration step or a combination thereof, preferably, wherein the first separation step comprises or consists of a centrifugation step.

4. The method according to any one of the preceding claims, wherein the inactivation treatment is an ultra high temperature (UHT) treatment, preferably carried out at a temperature of at least 120°C.

5. A method according to any one of the preceding claims, wherein: In step f., the pH of the biomass material is adjusted to pH 6.5 or below, such as 6.0 or below, 5.8 or below, preferably 5.5 or below, and / or wherein, In step f., the pH of the biomass material is adjusted to pH 4.0 or above, preferably 4.5 or above.

6. The method according to any one of the preceding claims, wherein the pH adjustment is performed by adding a C1-C5 organic acid, such as citric acid or propionic acid, preferably citric acid.

7. The method according to any one of the preceding claims, wherein the antioxidant is selected from ascorbic acid, ascorbyl stearate, tocopherol, rosemary extract, propyl gallate, quinones such as tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), β-carotene, β-apo-8'-carotenal, carotenic acid, ethyl ester, β-apo-8'-, citric acid, isopropyl citrate, thiodipropionic acid, dilauryl thiodipropionate, stearyl citrate and mixtures of two or more of such antioxidants.

8. The method according to any one of the preceding claims, further comprising a final step g. drying the biomass material to provide a powdered biomass material, optionally followed by a step h. granulating the powdered biomass material to provide a granulated biomass material.

9. The method according to any one of the preceding claims, wherein at least one methanotrophic bacterium is Methylococcus capsulatus.

10. The method according to any one of the preceding claims, wherein the fermentation step (a) comprises fermenting a mixture of methanotrophs and one or more heterotrophic bacteria, preferably wherein the heterotrophic bacteria are selected from the group consisting of Ralstonia sp., Bacillus brevis, Brevibacillusagri, Alcaligenes acidovorans, Aneurinibacillusdanicus and Bacillus firmus.

11. The method according to any one of the preceding claims, wherein steps e. and f. provide an oxidatively stable biomass material having an oxidative stability at least twice, preferably at least three times, greater than the oxidative stability of the same biomass material not subjected to steps e. and f.

12. A process according to any one of the preceding claims, further comprising the step of recycling the first liquid fraction from separation step (b) to fermentation step (a).

13. An oxidatively stable biomass material obtained from the fermentation of at least one methanotrophic bacterium, in the form of a dry powder or granules, comprising, by dry weight: - 60-75%, preferably 65-72% protein; - 1-10%, preferably 7-9% fatty acids; - 0.01-2% antioxidants; -in, When the biomass material is dissolved in water at a concentration of 100 to 400 g / L, the pH of the resulting solution is below 7.

0.

14. An oxidatively stable biomass material obtained from fermentation of at least one methanotrophic bacterium, wherein the oxidatively stable biomass material has an antioxidant stability at least two times, preferably at least three times, greater than the antioxidant stability of the same biomass material that has not been stabilized; wherein the stabilization comprises the following steps: - treating biomass material with antioxidants; and - Adjust the pH of the biomass material to 7.0 or below.

15. An aquafeed product comprising the oxidatively stable biomass material according to any one of claims 13-14, preferably 10-30% DM.

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