Acid-Stabilized Biomass Materials and Methods

Abstract

Provided is a method for producing a biomass material stabilized against oxidation. In particular, the method includes treating a concentrated biomass material with an antioxidant; and adjusting the pH of the concentrated biomass material to a pH of 7.0 or less. Through these steps, a synergistic stabilization of the biomass material is observed. Also provided are an oxidation-stabilized biomass material and an aquatic feed product containing the oxidation-stabilized biomass material.

Description

Technical Field

[0001] Provided is a method for producing a biomass material stabilized against oxidation. In particular, the method includes treating a concentrated biomass material with an antioxidant; and adjusting the pH of the concentrated biomass material to a pH of 7.0 or less. Through these steps, a synergistic stabilization of the biomass material is observed. Also provided are an oxidation-stabilized biomass material and an aquatic feed product containing the oxidation-stabilized biomass material.

Background Art

[0002] The methanotrophic bacterium Methylococcus capsulatus is a non-commensal bacterium that is ubiquitously found in nature. It metabolizes methane, for example, from natural gas into biomass, carbon dioxide, and water. Rich in protein, M. capsulatus can be used as a protein supplement in animal feed and is also interesting for human diets. For both animal and human diets, the fermentation of the bacterium as a protein source has the potential to contribute to meeting the global demand for edible proteins in a more environmentally friendly manner than the conventional protein manufacturing industry.

[0003] Typically, biomass materials contain fats and transition metals. The transition metals are present in the biomass material as a result of the fermentation medium used in the production of the biomass material. Attempts have been made to sequester the transition metals, such as by including one or more chelating agents, but reducing oxidation has not been proven.

[0004] Both fats and transition metals contribute to the formation of oxidation products, and as a result, the quality and shelf life of the product may be reduced. Furthermore, biomass products are typically heat-treated during production (e.g., spray drying and pelletizing), which is also thought to initiate or accelerate oxidation. Additional factors thought to accelerate oxidation include ease of contact with oxygen and light, and the moisture content of the biomass product.

Prior Art Documents

Non-Patent Literature

[0005]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Since fish do not eat oxidized feed, specific problems occur in fish feed. One object of the present invention is to provide a method and a biomass product with improved palatability.

Means for Solving the Problems

[0007] The present inventors have found that it is possible to protect biomass products against oxidation by adding antioxidants and changing or controlling the pH. The pH regulator and the antioxidant act synergistically to provide an increase in stability not seen when these components are used separately. In other words, treatment with only an antioxidant, only an acidity regulator, or an acidity regulator at the wrong pH does not show an increase in oxidation stability.

[0008] Therefore, in a first aspect, the present invention relates to a method for producing a biomass material stabilized against oxidation, said method comprising the following steps: a. Fermenting at least one methane-utilizing 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 an inactivation treatment on the concentrated biomass material; d. Homogenizing the concentrated biomass material if necessary; Here, steps c and d can be performed in any order. The method further includes the following additional steps: e. Treating the concentrated biomass material with an antioxidant after step b; and f. Adjusting the pH of the concentrated biomass material to a pH of 7.0 or less after step b.

[0009] There is also provided an oxidation-stabilized biomass material in the form of a dry powder or pellets, containing by dry weight: 60 - 75% protein; preferably 65 - 72% protein; 1 - 10% fatty acids; preferably 7 - 9% fatty acids; 0.01 - 2% antioxidant Provided that when the biomass material is dissolved in water at a concentration between 100 and 400 g / L, the resulting solution has a pH of 7.0 or less.

[0010] Furthermore, the oxidation-stabilized biomass material, the oxidation-stabilized biomass material has at least twice, preferably at least three times, the stability against oxidation of the same biomass material that has not been subjected to stabilization; the stabilization includes the following steps: Treating the biomass material with an antioxidant; and Adjusting the pH of the biomass material to a pH of 7.0 or less.

Advantages of the Invention

[0011] The stabilization treatment can be carried out in existing downstream manufacturing, but it is also possible to carry out in other process routes of fermentation-derived single-cell protein cultured using methanotrophic bacteria.

[0012] The above and other advantages of the present invention are presented in the following claims, figures, and examples.

Brief Description of the Drawings

[0013]

Figure 1

[0014] Detailed Disclosure Throughout this document, the abbreviation "DM" refers to "Dry Matter".

Mode for Carrying Out the Invention

[0015] Provided is a method for producing a biomass material thus stabilized against oxidation. The improved stability against oxidation is measured using an oxidation stability tester, such as the ML Oxipres manufactured by Mikrolab Aarhus A / S TM and the like.

[0016] The first step in this method is (a.) fermenting at least one methane-utilizing bacterium in a fermentation medium in the presence of a carbon source to provide a biomass material.

[0017] The biomass material is a single cell protein (SCP) product. It contains mostly protein (about 60%) and smaller amounts of RNA and DNA. When separated from the fermentation process, the biomass material is a water suspension. In the said water suspension, most of the solid components are cytoplasmic materials derived from methane-utilizing bacteria. Other components (such as proteins, nucleic acids, polysaccharides, lipids or other small molecules) may be dissolved or suspended in the aqueous phase.

[0018] At least one microorganism used in the fermentation process is a methane-utilizing bacterium, more preferably Methyococcus capsulatus. Therefore, the biomass is suitably Methyococcus capsulatus biomass.

[0019] The term "Methylococcus capsulatus" or "M. capsulatus" as used hereinabove can mean any strain of bacteria belonging to the M. capsulatus species. The strain may be a naturally occurring one or one developed in a laboratory such as a genetically engineered strain. The term "naturally occurring" means that the strain has not been genetically recombined using genetic engineering techniques. However, it may include natural modifications or changes in the genetic material, such as changes that occur randomly during replication, etc., compared to a reference strain. Preferably, the strain is a naturally occurring one. Similarly preferably, the strain is M. capsulatus (Bath), and more preferably M. capsulatus (Bath) identified as NCIMB 11132. However, it may also be M. capsulatus (Texas) or M. capsulatus (Aberdeen) or different M. capsulatus strains that are currently known or will be discovered or characterized in the future.

[0020] The methanotrophic bacterium may be provided in co-fermentation with one or more heterotrophic bacteria. The following heterotrophic bacteria may be particularly useful for co-fermentation with M. capsulatus; Ralstonia species; Bacillus brevis; Brevibacillus agri; Alcaligenes acidovorans; Anoxynatronobacterium danicus and Bacillus firmus. Suitable yeasts may be selected from the species of Saccharomyces and / or Candida. Preferred heterotrophic bacteria are selected from Alcaligenes acidovorans (NCIMB 13287), Anoxynatronobacterium danicus (NCIMB 13288) and Bacillus firmus (NCIMB 13289) and combinations thereof. The methanotrophic bacterium and / or the heterotrophic bacterium may be genetically recombined. In co-fermentation, M. capsulatus occupies 90 - 98%.

[0021] In fermentation engineering, the carbon source is converted by microorganisms into biomass material. Suitably, the carbon source includes methane and is, for example, natural gas, synthesis gas or biogas. In the fermentation process, the carbon source is dissolved in the fermentation medium. Fermentation is suitably carried out in a U-loop reactor as described in WO 2010 / 069313, which is incorporated herein by reference. A suitable fermentation medium is as described in, for example, WO 2018 / 158322, which is incorporated herein by reference. The fermentation process has, for example, a relatively low dry matter content of 5% or less.

[0022] Further details of the fermentation method are as described in WO 2020 / 245197 and WO 2020 / 249670, which are incorporated herein by reference.

[0023] 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 includes or consists of a centrifugation step, a membrane filtration step, or a combination thereof, preferably the first separation step includes or consists of a centrifugation step. The concentration of the biomass is carried out to provide a concentrated biomass material with a dry matter (DM) content between 5 and 25%, preferably between 10 and 20%.

[0024] When the first separation step is a centrifugation step, a first liquid fraction in the form of supernatant is provided. When the first separation step is a membrane filtration step, a first fraction in the form of filtrate permeate is provided.

[0025] The method further includes step (c) subjecting the concentrated biomass material to an inactivation treatment. This is because it is necessary to kill all living host organisms before the final product is achieved. The inactivation treatment may include one or more ultra-high temperature (UHT) treatments, ultraviolet radiation or treatments using sterile filtration, and is preferably an ultra-high temperature (UHT) treatment. Preferably, the UHT treatment is carried out at a temperature of at least 120°C, preferably between 120 and 135°C. Suitably, the UHT treatment is carried out over 5 and 60 minutes.

[0026] To improve the uniformity of the concentrated biomass material, this method may include the optional step (d) of homogenizing the concentrated biomass material. Homogenization is typically carried out at a pressure between 5 and 900 bar in a homogenizer vessel. During homogenization, large particles in the concentrated biomass material are broken down, giving a more uniform distribution.

[0027] The steps of inactivation and homogenization (steps c. and d.) can be carried out in any order. Preferably, step c. is carried out before step d.

[0028] At this point in the method, the concentrated biomass material typically has a pH around 6.8 - 7.0.

[0029] The method according to the invention includes the following additional steps: e. treating the concentrated biomass material with at least one antioxidant after step b; and f. adjusting the pH of the concentrated biomass material to a pH of 7.0 or less after step b.

[0030] As mentioned above, both of these steps are carried out after step b, i.e., the separation step. Treatment with an antioxidant and pH adjustment act synergistically to improve the resistance of the biomass material to oxidation. In other words, simply adding only an antioxidant or only a pH regulator is insufficient.

[0031] In one aspect, steps e. and f. are carried out simultaneously. In another aspect, step f. is carried out before step e. Steps e. and f. may be carried out after step d, or steps e. and f. may be carried out before step d. Overall, the preferred method includes steps a - f in sequence.

[0032] The following steps: e. treating the concentrated biomass material with at least one antioxidant after step b; and f. After step b, adjust the pH of the concentrated biomass material to a pH of 7.0 or less. This may be done in the same vessel, suitably with stirring for at least 30 minutes. The temperature of the biomass material in the vessel can be that obtained from the previous step (e.g., 20 - 40 °C) or the biomass material in the vessel can be actively cooled to 7 - 10 °C.

[0033] In a further aspect, both step c and step d are carried out, and step c is carried out before step d.

[0034] The method may further include a step of water evaporation between step b and step c, between step b and step d, and / or between step c and step d. In such an evaporation step, typically a dry matter content of 20 - 40%, preferably 25 - 35% is obtained.

[0035] Step f is suitably carried out by adding an acid or a buffer to the concentrated biomass material to reach the required pH. Measurement of the pH of the biomass material is within the competence of the person skilled in the art. Preferably in step f, the pH of the biomass material is adjusted to a pH of 6.5 or less, e.g., 6.0 or less, 5.8 or less, preferably 5.5 or less. The adjustment of the pH can be carried out by adding an inorganic or organic acid, preferably an organic acid. In one aspect, the adjustment of the pH is carried out by adding a C1 - C5 organic acid, e.g., citric acid or propionic acid, preferably citric acid. Citric acid is preferred due to its polyacid functionality and the possibility of creating a stable buffer solution. Suitably, in step f, the pH of the biomass material is adjusted to a pH of 4.0 or more, preferably 4.5 or more.

[0036] The present invention may use various 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, beta-apo-8'-carotenal, carotenic acid, ethyl ester, beta-apo-8'-, citric acid, isopropyl citrate, thiodipropionic acid, dilauryl thiodipropionate, and stearyl citrate. Mixtures of two or more such antioxidants are also possible. 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, beta-apo-8'-carotenal, carotenic acid, ethyl ester, beta-apo-8'-, thiodipropionic acid and dilauryl thiodipropionate. Tocopherol-containing antioxidants are most preferred as they are natural antioxidants.

[0037] In one aspect, the method further comprises a final step g. of 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. of pelletizing the powdered biomass material to provide a pelletized biomass material.

[0038] As described above, the method of the present invention provides an oxidation-stabilized biomass material. Preferably, the oxidation-stabilized biomass material has at least twice, preferably at least three times, the stability against oxidation of the same biomass material that has not been subjected to steps e. and f.

[0039] By the method of the present invention, an oxidation-stabilized biomass material in the form of a dry powder or pellets may be produced. The material, on a dry weight basis, comprises: 60 - 75%, preferably 65 - 72% protein; 1 - 10%, preferably 7 - 9% fatty acids; 0.01 to 2% antioxidant However, when the biomass material is dissolved in water at a concentration between 100 and 400 g / L, the resulting solution has a pH of 7.0 or less.

[0040] There is also provided an oxidation-stabilized biomass material, which has at least twice, preferably at least three times, the stability against oxidation of the same biomass material not subjected to stabilization; said stabilization includes the following steps: Treating the biomass material with an antioxidant; and Adjusting the pH of the biomass material to a pH of 7.0 or less.

[0041] The method and the oxidation-stabilized biomass material are useful in an aquatic environment (i.e., as fish feed), where the problem of oxidation is highly relevant. Thus, the present invention provides an aquatic feed product comprising the oxidation-stabilized biomass material described so far, preferably at 10 to 30% DM.

[0042] Detailed description M. capsulatus biomass can be prepared by the method described by Larsen and Jorgensen, Appl. Microbiol. Biotechnol., 1996. M. capsulatus, for example M. capsulatus bath (NCIMB 11132), grows in a bioreactor in a suitable medium using methane as a carbon source.

[0043] The downstream manufacturing method for biomass derived from fermentation based on the culture of methanotrophic strains is shown in Figure 1. As can be seen from steps 1 to 3 of Figure 1, said downstream manufacturing concentrates the biomass from 1 to 2% DM to approximately 10 to 20% DM. The product is then heat-treated (step 4) at 120 to 135 °C and then homogenized (step 5) at 6 to 900 bar. Optionally, the product can then be spray-dried (step 7) and pelletized (step 8).

[0044] In the present invention, additional processing steps are carried out as seen in Figure 1, where in that case, the biomass after concentration (step 3) is treated with an antioxidant and the pH is changed / controlled somewhere in the existing method. There are five possible manufacturing routes for the treatment using an antioxidant and pH. All five possibilities are illustrated in Figure 1. The most commonly used downstream method is from step 1 to 8, where in that case, the treatment can be carried out in a storage tank in step 6 before spray drying.

[0045] The adjustment of pH is typically carried out before the addition of the antioxidant. The pH is adjusted using an acid, where in that case, citric acid is preferred but propionic acid can be an alternative. The pH is adjusted to 7.0 or less, preferably 6.0 or less. Addition of antioxidant: The antioxidant is weighed and added after the adjustment of pH in an amount of 0.05 - 3 g / l. Stirring of the tank used for the treatment is usually required. Cooling is optional.

[0046] The present invention provides single cell protein (SCP) resulting from fermentation cultured using methanotrophic bacteria against oxidation when used, for example, in fish feed formulations.

[0047] Description of the drawings Figure 1 depicts five manufacturing routes available in the present invention.

[0048] The first method route includes the following: 1. Fermenter of 1.1 - 2% DM 2. Separator for concentrating the biomass to 10 - 20% DM 3. Concentrated biomass 4a. UHT treatment at 120 - 135 °C 5a. The biomass is homogenized at 5 - 900 bar 6a. Balance tank to which a pH regulator and an antioxidant for treating the biomass (adjusting the pH to 7.0 or less and adding an antioxidant in the range of 0.05 - 3 g / l) are added 7. The biomass is spray dried - powder product P1 is produced. 8 The powder is pelletized - pellet product P2 is produced.

[0049] The second process route includes the following: 1. A fermenter with 1.1 - 2% DM 2. A separator for concentrating the biomass to 10 - 20% DM 3. Concentrated biomass 4. UHT treatment at 120 - 135 °C 5. A balance tank to which a pH regulator and an antioxidant for treating the biomass (adjusting the pH to 7.0 or less and adding an antioxidant in the range of 0.05 - 3 g / l) are added 6. The biomass is homogenized at 5 - 900 bar 7. The biomass is spray - dried - Powder product P1 is produced 8. The powder is pelletized - Pellet product P2 is produced

[0050] The third process route includes the following: 1. A fermenter with 1.1 - 2% DM 2. A separator for concentrating the biomass to 10 - 20% DM 3. Concentrated biomass 4b. UHT treatment at 120 - 135 °C 5b. Evaporation of the biomass to a concentration of 25 - 35% DM 6b. The biomass is homogenized at 5 - 900 bar 6c. A balance tank to which a pH regulator and an antioxidant for treating the biomass (adjusting the pH to 7.0 or less and adding an antioxidant in the range of 0.05 - 3 g / l) are added 7. The biomass is spray - dried - Powder product P1 is produced 8 The powder is pelletized - Pellet product P2 is produced

[0051] The fourth process route includes the following: 1. A fermenter with 1.1 - 2% DM 2. A separator for concentrating the biomass to 10 - 20% DM 3. Concentrated biomass 4c. The biomass is homogenized at 5 - 900 bar 5c. UHT treatment at 120 - 135 °C Balance tank to which a pH adjuster and an antioxidant for treating biomass (adjusting the pH to 7.0 or less and adding an antioxidant in the range of 0.05 to 3 g / l) are added 7. The biomass is spray-dried - Powder product P1 is produced 8 The powder is pelletized - Pellet product P2 is produced

[0052] The fifth process route includes the following: 1. Fermenter with 1 - 2% DM 2. Separator for concentrating biomass to 10 - 20% DM 3. Concentrated biomass 4d. The biomass is homogenized at 5 - 900 bar 5d. Balance tank to which a pH adjuster and an antioxidant for treating biomass (adjusting the pH to 7.0 or less and adding an antioxidant in the range of 0.05 to 3 g / l) are added 6d. The biomass is inactivated, for example, by ultraviolet radiation or filtration 7. The biomass is spray-dried - Powder product P1 is produced 8 The powder is pelletized - Pellet product P2 is produced

[0053] ML Oxipres TM Illustrated procedure for measuring oxidative stability using

[0054] Procedure Select an appropriate oxidation temperature (e.g., 80 °C). Start the heater 20 minutes before the start of the experiment Weigh an appropriate amount of the sample into each glass container. (An amount containing 3 - 5 g of fat often gives reasonable results). Place a glass lid on top of the container Place the glass container inside the pressure vessel (bomb) Check that the O-ring and groove are clean. Fix the top of the pressure vessel and tighten it by hand Connect the pressure vessel to the filling station a. Close the valve on the filling station b. Close the regulator outlet valve c. Open the main cylinder valve (gradually). d. Adjust the outlet pressure to 5 bar (70 psig) in the regulator. e. Open the pressure vessel valve. 7. a. Open the regulator outlet valve. b. Turn the filling station valve to FILL. c. Turn the valve to VENT to flow the pressure vessel with atmospheric air. d. Repeat b and c to repeat the flow (repeating 3 times results in a nitrogen content of 1% or less). 8. Now fill the pressure vessel using oxygen. Reach 5 bar (70 psig) or less using a pressure control cylinder. Read the pressure of the pressure vessel on the display on the control device. 9. Close the filling station valve (OFF) and then close the pressure vessel valve. 10. Monitor the pressure for a few minutes to see if the pressure vessel was properly tightened. 11. If tightened, turn the filling station valve to VENT and disconnect the filling tube from the pressure vessel. 12. Place the pressure vessel in the block heater and start the test. 13. Don't forget to shut off the cylinder main valve. 14. After the test, the valve is opened. Exhaust can be done through an odor trap (carbon filter) or in a fume cupboard to reduce any possible odors.

[0055] Evaluation of Results As a result of oxygen consumption, the pressure in the bomb decreases. (Initially the pressure rises due to heating). The signal from the pressure transducer is shown on the display. The signal is recorded as a function of time.

[0056] The sample is characterized by an induction time. The induction time (IP, unit of time) is found by plotting the signal as a function of time and drawing tangents T1 and T2 to the curve. The time from START to the intersection point is the IP. START is when the pressure vessel is placed in the block heater. The induction time thus becomes the time elapsed between placing the pressure vessel in the block heater and the break-point at a predetermined temperature (e.g., 80 °C).

Example

[0057] Experimental results: At the beginning of the study, peroxides (PV) and thiobarbituric acid (TBA) were focused on as oxidation parameters. Later, Oxipres, i.e., oxidation stability, was used by default with improved reliability.

[0058] The first test (sample A, without pH regulator or antioxidant) showed a reference stability of 17.5 hours (i.e., low stability). The addition of antioxidant alone did not provide a significant increase in stability (sample B).

[0059] When the product was treated using different combinations of pH regulators and antioxidants (samples C - F), stability up to 75 hours was observed. The procedure was tested on a larger scale (sample E), in which case a stability of 61.3 hours was observed. It was suggested that the product is non-homogenized and can be processed and maintain oxidative stability in different types of biomass products. In sample F, testing of another antioxidant from Vitablend was successful and thus the treatment does not depend on one brand. Samples that adsorb oxygen quickly have low resistance to oxidation (corresponding to low times in the Oxipres test).

[0060]

Table 1

Explanation of symbols

[0061] 1 Fermenter 2 Separator 3 Recovery (Pellet) 4 UHT 120 - 135 degrees 5 Balance Tank 6 Homogenisator 7 Spray Drying 8 Pelletizer P1 Powder P2 Pellet

[0062] Provide the following list of numbered embodiments: Embodiment 1. A method for producing a biomass material stabilized against oxidation, the method comprising the following steps: a. Fermenting at least one methane - assimilating 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 an inactivation treatment on the concentrated biomass material; d. Optionally, homogenizing the concentrated biomass material; where steps c. and d. can be performed in any order; The method further comprises the following additional steps: e. Treating the concentrated biomass material with at least one antioxidant after step b; and f. Adjusting the pH of the concentrated biomass material to a pH of 7.0 or less after step b.

[0063] Embodiment 2. The method according to Embodiment 1, wherein steps e. and f. are performed simultaneously.

[0064] Embodiment 3. The method according to Embodiment 1, wherein step f. is performed after step e.

[0065] Embodiment 4. The method according to any one of the preceding embodiments, wherein steps e. and f. are performed after step d., or steps e. and f. are performed before step d.

[0066] Aspect 5. The method according to any one of the preceding aspects, wherein the first separation step includes a centrifugation step, a membrane filtration step, or a combination thereof, preferably the first separation step includes or consists of a centrifugation step.

[0067] Aspect 6. The method according to any one of the preceding aspects, wherein the inactivation treatment is preferably an ultra-high temperature (UHT) treatment at a temperature of at least 120°C.

[0068] Aspect 7. The method according to any one of the preceding aspects, wherein step c. is performed before step d.

[0069] 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 a pH of 6.5 or less, for example 6.0 or less, 5.8 or less, preferably 5.5 or less.

[0070] 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 a pH of 4.0 or more, preferably 4.5 or more.

[0071] 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.

[0072] Aspect 11. The antioxidant is selected from the group consisting of ascorbic acid, ascorbyl stearate, tocopherol, rosemary extract, propyl gallate, quinones such as tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), β-carotene, beta-apo-8'-carotenal, carotenic acid, ethyl ester, beta-apo-8'-, citric acid, isopropyl citrate, thiodipropionic acid, dilauryl thiodipropionate, stearyl citrate and mixtures of two or more such antioxidants, according to any one of the preceding aspects.

[0073] Aspect 12. The method according to any one of the preceding aspects, further comprising a step of water evaporation between step b and step c, between step b and step d, and / or between step c and step d.

[0074] Aspect 13. The method according to any one of the preceding aspects, further comprising a final step g of drying the biomass material to provide a powdered biomass material.

[0075] Aspect 14. The method according to aspect 13, further comprising a step h of pelletizing the powdered biomass material to provide a pelletized biomass material.

[0076] Aspect 15. The method according to any one of the preceding aspects, wherein the at least one methane-utilizing bacterium is Methyococcus capsulatus.

[0077] Aspect 16. The method according to any one of the preceding aspects, wherein the biomass is Methyococcus capsulatus biomass.

[0078] Aspect 17. The method according to any one of the preceding aspects, wherein the fermentation step (a) comprises the fermentation of a mixture of methanotrophic bacteria and one or more heterotrophic bacteria.

[0079] Aspect 18. The method according to aspect 17, wherein the heterotrophic bacteria are selected from the group consisting of Ralstonia species; Bacillus brevis; Brevibacillus agri; Alcaligenes acidorbans; Anaerolinobacillus danicus and Bacillus firmus.

[0080] Aspect 19. The method according to any one of the preceding aspects, wherein the carbon source comprises methane and is, for example, natural gas or biogas.

[0081] Aspect 20. The method according to any one of the preceding aspects, wherein steps e and f provide an oxidation-stabilized biomass material having at least twice, preferably at least three times, the stability to oxidation of the same biomass material not subjected to steps e and f.

[0082] Aspect 21. The method according to any one of the preceding aspects, further comprising a step of recycling the first liquid fraction from the separation step (b) to the fermentation step (a).

[0083] Aspect 22. An oxidation-stabilized biomass material in the form of a dry powder or pellet, obtained from the fermentation of at least one methanotrophic bacterium, comprising the following by dry weight: 60 to 75%, preferably 65 to 72% protein; 1 to 10%, preferably 7 to 9% fatty acids; 0.01 to 2% antioxidant Provided that when the biomass material is dissolved in water at a concentration between 100 and 400 g / L, the resulting solution has a pH of 7.0 or less.

[0084] Aspect 23. An oxidation-stabilized biomass material obtained from the fermentation of at least one methanotrophic bacterium, wherein the oxidation-stabilized biomass material has at least twice, preferably at least three times, the stability to oxidation of the same biomass material that has not been subjected to stabilization; the stabilization includes the following steps: Treating the biomass material with an antioxidant; and Adjusting the pH of the biomass material to a pH of 7.0 or less.

[0085] Aspect 24. An aquatic feed product comprising the oxidation-stabilized biomass material according to any one of Aspects 22 to 23, preferably at 10 to 30% DM.

Claims

1. A method for producing biomass material stabilized against oxidation, comprising the following steps: a. To provide biomass material by fermenting at least one methane-utilizing bacterium in a fermentation medium in the presence of a carbon source containing methane; b. In the first separation step, the biomass material is separated to provide concentrated biomass material and a first liquid fraction; c. Inactivating concentrated biomass material; d. If necessary, homogenize the concentrated biomass material; Here, steps c. and d. can be performed in any order. The above method includes the following additional steps: e. Process b. Afterwards, treat the concentrated biomass material with at least one antioxidant; and f. After step b., adjust the pH of the concentrated biomass material to 7.0 or lower.

2. The aforementioned - Are steps e. and f. performed simultaneously? - Whether process f. is performed after process e. - Is process c performed before process d? - Steps e. and f. are performed after step d., or - Steps e. and f. are performed before step d. The method according to claim 1.

3. The method according to claim 1 or 2, wherein the first separation step includes a centrifugal separation step, a membrane filtration step, or a combination thereof.

4. The method according to claim 1 or 2, wherein the deactivation treatment is an ultra-high temperature (UHT) treatment at a temperature of at least 120°C.

5. The method according to claim 1 or 2, wherein in step f, the pH of the biomass material is adjusted to be 6.5 or less, and / or in step f, the pH of the biomass material is adjusted to be 4.0 or more.

6. The pH adjustment mentioned above is C １ ~C ５ The method according to claim 1 or 2, which is carried out by adding an organic acid.

7. The method according to claim 1 or 2, wherein the antioxidant is selected from the group consisting of ascorbic acid, ascorbyl stearate, tocopherol, rosemary extract, propyl gallate, quinone, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), β-carotene, beta-apo-8'-carotenal, carotenic acid, ethyl ester, beta-apo-8'-, citric acid, isopropyl citrate, thiodipropionic acid, dilauryl thiodipropionate, stearyl citrate, and mixtures of two or more such antioxidants.

8. The method according to claim 1 or 2, further comprising a final step g. of drying biomass material to provide powdered biomass material, and a subsequent step h. of pelletizing the powdered biomass material to provide pelletized biomass material.

9. The method according to claim 1 or 2, wherein the at least one methane-utilizing bacterium is Methylococcus capsulatus.

10. The method according to claim 1 or 2, wherein the fermentation step (a) comprises fermentation of a mixture of methane-producing bacteria and one or more heterotrophic bacteria, the heterotrophic bacteria being selected from the group consisting of Ralstonia species; Bacillus brevis; Brevibacillus agri; Alcaligenes acidoborans; Aneurinibacillus danicus and Bacillus farmus.

11. The method according to claim 1 or 2, wherein steps e. and f. provide an oxidation-stabilized biomass material having at least twice the oxidation stability of the same biomass material not subjected to steps e. and f.

12. The method according to claim 1 or 2, further comprising the step of reusing the first liquid fraction from the separation step (b) to the fermentation step (a).

13. Oxidation-stabilized biomass material obtained from the fermentation of at least one methane-utilizing bacterium, in the form of a dry powder or pellet, comprising the following by dry weight: 60-75% protein; 1-10% fatty acids; 0.01-2% antioxidant However, when the biomass material is dissolved in water at a concentration between 100 and 400 g / L, the resulting solution has a pH of 7.0 or less.

14. An oxidation-stabilized biomass material obtained from the fermentation of at least one methane-utilizing bacterium, wherein the oxidation-stabilized biomass material has at least twice the oxidation stability of the same biomass material that was not subjected to stabilization; the stabilization includes the following steps: Treating biomass materials with antioxidants; and 7. Adjust the pH of the biomass material to a pH of 0 or lower.

15. An aquatic feed product comprising 10-30% DM of the oxidation-stabilized biomass material according to claim 13 or 14.

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