Process for preparing microalgae-based haem proteins for food use

Abstract

The present invention relates to a process for preparing haem proteins in microalgae, as well as to the use of said obtained haem proteins for coloring a food, preferably a vegetal steak.

Description

Process for the production of heme proteins based on microalgae for use in food

[0001] The present patent application relates to a process for producing heme proteins in microalgae, as well as the use of said heme proteins obtained for coloring a food, preferably a vegetable steak. Prior art

[0002] Meat products occupy an important place in our diet. They are consumed in many different forms, such as whole muscles or chopped cuts of meat that are baked, grilled, smoked, dried, or fermented in the form of burgers, patties, sausages, meatballs, or nuggets.

[0003] More and more consumers are looking to reduce or completely eliminate animal products from their diets, particularly red meat. They are turning to plant-based meat substitutes for a variety of reasons, including health concerns, environmental concerns, sustainability, and animal welfare.

[0004] For a regular meat consumer to choose to consume meat substitutes, it is essential that these substitutes mimic processed meat products in terms of texture, organoleptic properties such as appearance, flavor, and mouthfeel.

[0005] Various functional ingredients in meat substitutes are needed to mimic the taste, flavor, texture, mouthfeel, and appearance of meat products.

[0006] There are now processes / compositions that allow us to obtain plant-based alternatives to meat, mainly composed of soy and legumes. However, these formulas often contain a large number of ingredients, including synthetic food additives, and present color problems. Indeed, few plant-based substitutes manage to imitate the color of red meat with its characteristic browning when cooked.

[0007] Patent CN111449203 (A) describes a plant-based meat substitute composition incorporating soy, white beans, and red beans as the main components, plus 8 other ingredients. No mention of color is made.

[0008] Similarly, patent CN110292098 (A) mentions the use of soy, lotus leaf, peanuts, wheat protein and starch in the composition of its plant-based meat. Again, the concept of color is not addressed here.

[0009] In order to give plant-based meat a color as similar as possible to that of animal meat, different ingredients can be used, such as caramel, malt extract, or beet juice (Asgar et al. 2010). However, these ingredients do not allow the characteristic change in color of meat during cooking. Indeed, to mimic this browning linked to the oxidation of iron in the myoglobin present in the animal's muscles, it is advisable to use heme proteins of plant origin with a structure and behavior similar to myoglobin.

[0010] Thus, patent US2019292217 (A1) describes the use of transgenic plants overexpressing the heme protein biosynthesis pathway.

[0011] Similarly, Triton has filed patents related to the production of heme proteins such as protoheme or protoporphyrin IX by algae. Patent CN111655042 (A) presents a technique for obtaining algae overexpressing protoporphyrin IX through genetic engineering.

[0012] However, most of the plant-based meat compositions detailed in the previously cited patents do not address the issue of color, and the natural options available on the market do not achieve the characteristic color change (browning) of the meat during cooking.

[0013] In order to offer a natural ingredient giving plant-based meat a color similar to that of animal meat, changing from red to brown after heating, the Applicant has developed a technique allowing the production of microalgae naturally rich in heme proteins (e.g. cytochrome c), without recourse to genetic modifications (GMOs).

[0014] This process also has the advantage of being natural and therefore better accepted by the consumer. Description of the invention

[0015] Thus, according to a first aspect, the invention relates to a method for producing heme proteins in microalgae.

[0016] According to one embodiment, this method for producing hemin proteins in microalgae comprises the steps of:Acclimating microalgal strains;Continuously culturing said strains acclimated in step a. in a turbidostat under conditions of oxidative stress for the cells of said strains, preferably by saline, light or metal stress, optionally associated with an excess of iron;Harvesting the microalgal cells obtained in step b. in order to remove the culture medium by flocculation, flotation, coagulation, filtration or centrifugation, preferably by centrifugation;Optionally, preserving the microalgal cells harvested in step c. at a temperature between -20°C and -90°C, preferably at -80°C, until extraction step e.;Extracting the cellular compounds by membrane rupture of the cells obtained in step c. or d. ;Solid-liquid separation of the cells obtained in step e., preferably by filtration, decantation or centrifugation, more preferably centrifugation;Separation of the solid fraction comprising the proteins obtained in step f., preferably by ammonium sulfate precipitation, by size exclusion chromatography, by ion exchange chromatography, by filtration, even more preferably by ammonium sulfate precipitation.Centrifugation of the precipitate obtained in step g. to obtain a heme protein precipitate;Recovery of the centrifugation pellet containing the heme protein precipitate and solubilization of the heme protein precipitate obtained in step h. in an aqueous phase;Obtaining heme proteins in aqueous solution.

[0017] This process, via heterotrophic culture in a situation of oxidative stress in a medium optionally concentrated in iron, allows to favor the accumulation of heme proteins in the algae. Indeed, oxidative stress provokes a defense response of the algae which produces a greater quantity of heme proteins using the available iron of the medium. The algae thus cultivated will produce 5% to 30% more heme proteins than an algae cultivated in classic conditions of photoautotrophy and / or heterotrophy. The microalgae strains incapable of surviving these stress conditions via the production of heme proteins will be eliminated as the culture continues in turbidostat, thus favoring a line of microalgae with a high content of heme proteins which can be preserved in order to produce a natural food ingredient used to color vegetable meat.

[0018] By "acclimatization of microalgae strains" is meant according to the invention that said strains are conditioned, in an environment, so that they adapt there.

[0019] Preferably, the acclimatization step a. is carried out, preferably in flasks, at a temperature between 15°C and 35°C, preferably between 25°C and 30°C, for a duration between 3 and 6 days.

[0020] By "continuous culture" is meant according to the invention that the system is an open system in which the population of cells is constantly maintained in a medium balanced in nutrients and cell concentration.

[0021] By "cellular compounds" is meant all the elements contained in the cell or in its membranes, including heme proteins.

[0022] Step b. of culturing in a turbidostat under oxidative stress conditions makes it possible to increase the production of heme in the microalgae.

[0023] During this stage, due to the rapidity of the microalgae life cycle, it is possible to rapidly accumulate mutations during the microorganism's natural cell division by mitosis. This makes it possible to obtain several hundred generations of microalgae in a short period of time.

[0024] A turbidostat is defined as a continuous culture bioreactor with continuous turbidity measurement. This device allows for the continuous culture or fermentation of microorganisms, in this case microalgae.

[0025] Turbidity is measured as optical density every 6 minutes using a microcomputer and an optical sensor. When the signal falls below a preset value, a peristaltic pump is activated to regulate the algae density to approximately 25% of the maximum yield density.

[0026] The culture in the turbidostat is carried out in the presence of a culture medium allowing the development of microalgae. A person skilled in the art is able to determine such a culture medium.

[0027] An example of such a culture medium is the f / 2 culture medium, comprising:Na NO3 (75.0 g / L dH2O)Na H2 PO4·H2O (5.0 g / L dH2O)Na2 SiO3·9H2O (30.0 g / L dH2O)Na2H2EDTA (4.36 g / L)FeCl3 6H2O (3.15 g / L)MnCl2 4H2O (180 mg / L)ZnSO4 (22 mg / L)CoCl2 6H2O (10 mg / L)CuSO4 5H2O (9.8 mg / L)Na2MoO4 2H2O (6.3 mg / L)Thiamine HCl (200 mg / L)Cyanocobalamin (10 mg / L)Biotin (100 mg / L)Filtered seawater

[0028] This medium is autoclaved after adding all the components.

[0029] Another example of such a culture medium is the “Knop's solutions” medium which comprises in g / L: 1.25, KNO3; 1.25 KH2PO4; 2.5 MgSO47H2O; 0.30 Na citrate; 0.004 Fe2(SO4)3; 0.0029 H3BO3; 0.0018, MnCl24H2O; 0.00022 ZnSO47H2O; 0.000080 CuS045H2O; 0.000018 MoO3

[0030] Oxidative stress is a type of aggression against cell constituents, which appears when reactive oxygen species (or free radicals) and / or oxidizing reactive oxygen and nitrogen species enter the cell or are formed there; these molecules are unstable and very cytotoxic because they "oxidize" other molecules by removing an electron from them, which in turn makes them unstable.

[0031] This applied oxidative stress will vary depending on the species of algae cultivated.

[0032] It can thus take the form of: Salt stress, comprising the use of a salt concentration of between 30 and 500 mM NaCl for 1 to 5 days; or Light stress, by intermittent exposure to 30 and 500 µmol of photons / m2 / s for 30 to 120 minutes, or prolonged exposure to a high light intensity of the order of 200 – 500 µmol of photons / m2 / s for 0.5h to 72h; or Metal stress, by adding copper in concentrations of the order of 5 to 500 µM or concentrations of the order of 5 to 500 µg / L for 1 to 5 days;

[0033] Salt stress is not used for halotolerant species such as Dunaliella.

[0034] According to one embodiment, the saline stress is carried out by adding 300 mM NaCl.

[0035] Oxidative stress may optionally be associated or not with an excess of iron in the culture medium with a concentration of 0.5 to 40 mg / L.

[0036] Iron can be found in different forms: ferric chloride, ferrous chloride, ferric EDTA, ferrous EDTA, ferric ammonium sulfate and ferrous ammonium sulfate.

[0037] Preferably, it may be FeCl3 6H2O at 5 g / L.

[0038] For the purposes of the invention, “harvesting microalgae” means that the culture medium is removed in order to isolate the microalgae obtained at this stage.

[0039] For the purposes of the invention, "flocculation" means the physicochemical process during which materials suspended in a liquid agglomerate to form larger, generally very porous particles, called flocs.

[0040] For the purposes of the invention, "flotation" means this separation process based on the differences in hydrophobicity of the surfaces of the particles to be separated.

[0041] For the purposes of the invention, "coagulation" means this separation process based on the destabilization of particles by the addition of a chemical reagent.

[0042] For the purposes of the invention, "filtration" means the technique which makes it possible to separate the constituents of a mixture using a filter which makes it possible to retain particles larger than the pores of the filter.

[0043] For the purposes of the invention, "centrifugation" means this technique which, through the action of centrifugal force, separates the phases of a mixture. This mixture is driven into a very rapid rotational movement, and the heavier particles are then pushed towards the walls of the container under the action of centrifugal force, while the lighter particles and liquids remain on the surface (supernatant).

[0044] Preferably, step c. of harvesting the microalgal cells obtained in step b. is carried out by centrifugation lasting 5 to 20 min at a rotation speed of between 1000 and 2500 G and at a temperature of between 10°C and 20°C, in order to remove the culture medium.

[0045] According to one embodiment, the method comprises a step d. of preserving the microalgae cells harvested in step c. at a temperature between -20°C and -90°C, preferably at -80°C, while awaiting step e. of extraction.

[0046] For the purposes of the invention, "membrane rupture" means the lysis of cells in order to obtain all of their membrane and intracellular contents.

[0047] Preferably, step e. of extracting the cellular compounds is carried out by membrane disruption of the cells obtained in step c. or d. with a biomass / solvent ratio of between 1:5 and 1:50 (v / v) in an aqueous solvent with a pH of between 6 and 12 at a temperature of between 5°C and 45°C: Either by using ultrasound at a frequency of between 10 and 40 kHz, with a power of 50-1000 W for a duration of between 5 and 60 minutes; or Either by high pressure homogenization (HPH), from 1 to 4 cycles at a pressure of between 800 bars and 2500 bars; or by ball milling, with balls of diameter 0.4 to 2 mm, filling the extraction chamber between 60 and 90%, at a rotation speed of 1000 to 4000 rpm for 15 to 90 minutes.

[0048] For the purposes of the invention, the term "use of ultrasound" means that the extraction is carried out by applying ultrasonic waves to the solution which propagate through the liquid medium.

[0049] For the purposes of the invention, "high pressure homogenization" means that the mixing is carried out in a device capable, by applying significant pressure, of creating microscopic particles enabling substances to be mixed.

[0050] For the purposes of the invention, the term "ball milling" means the use of a device consisting of a horizontal drum rotated by a motor. The drum is partially filled with the product to be ground to which the grinding elements (metal balls, for example) are added.

[0051] Preferably, step f. of solid-liquid separation of the cells obtained in step e. is carried out by centrifugation at a speed of between 2,000 and 10,000 rpm for 5 to 30 min at a temperature of between 10°C and 20°C.

[0052] For the purposes of the invention, the term "solid-liquid separation" means this process for separating the solid phase and the liquid phase of a mixture.

[0053] “Protein precipitate” means the solid mass of proteins obtained following step f. of solid-liquid separation.

[0054] For the purposes of the invention, "ammonium sulfate precipitation" means this method for purifying proteins by precipitation. Due to its ionic nature, ammonium sulfate is very soluble in water and can therefore "precipitate" proteins by precipitation.

[0055] For the purposes of the invention, "steric exclusion chromatography" means this liquid chromatography method for separating macromolecules according to their hydrodynamic volume.

[0056] For the purposes of the invention, "ion exchange chromatography" or "ion chromatography" means this technique which separates molecules according to their respective charged groups.

[0057] Preferably, step g. of separation of the solid fraction comprising the proteins is carried out by precipitation with ammonium sulfate, with a saturation of 40% to 80%.

[0058] Preferably, step h. of centrifugation of the precipitate obtained in step g. lasts 5 to 30 minutes, at a rotation speed of between 2,000 and 10,000 rpm at a temperature of 4-15°C, making it possible to obtain a protein precipitate.

[0059] For the purposes of the invention, the term "centrifugation pellet" means what is found at the bottom of the tube once centrifugation is complete, and which therefore includes the elements which formed the protein precipitate.

[0060] Preferably, step i. of recovering the centrifugation pellet containing the protein precipitate and solubilizing the protein precipitate obtained in step h. is carried out in an aqueous phase with a pH between 6.5 and 12.

[0061] By "solubilization of the precipitate" is meant within the meaning of the invention that the precipitate obtained is brought into contact with water in order to regain a solution form of the hemin proteins.

[0062] Preferably, in order to obtain the hemin proteins in powder form, said method according to the invention further comprises: Drying the solution of hemin proteins obtained in step j, by freeze-drying, atomization, low temperature drying or pressure drying, preferably by atomization or freeze-drying. Obtaining hemin proteins in powder form.

[0063] For the purposes of the invention, "drying" means the action of removing the aqueous phase from the solution, in order to obtain the target compounds in powder form.

[0064] By "in powder form" is meant that the heme proteins obtained are in solid form, made up of very fine particles.

[0065] According to one embodiment, the method according to the invention comprises the steps of:Acclimatization of microalgae strains, preferably in flasks, at a temperature between 15°C and 35°C, preferably between 25°C and 30°C, for a duration between 3 and 6 days;Continuous cultivation of said strains acclimatized in step a.in a turbidostat under conditions of oxidative stress for the cells of said stems, said oxidative stress being chosen from:Salt stress, comprising the use of a salt concentration of between 30 and 500 mM of NaCl for 1 to 5 days; orLight stress, by intermittent exposure to 30 and 500 µmol of photons / m2 / s for 30 to 120 minutes, or prolonged exposure to a high light intensity of the order of 200 – 500 µmol of photons / m2 / s for 0.5h to 72h; orMetal stress, by adding copper in concentrations of the order of 5 to 500 µM or concentrations of the order of 5 to 500 µg / L for 1 to 5 days; said stress can optionally be associated with an excess of iron in the culture medium with a concentration of 0.5 to 40 mg / L.Harvesting of the microalgae cells obtained in step b.by centrifugation for 5 to 20 min at a rotation speed between 1000 and 2500 G and at a temperature between 10°C and 20°C, in order to remove the culture medium;Optionally, preservation of the microalgal cells harvested in step c. at a temperature between -20°C and -90°C, preferably at -80°C, while awaiting extraction step e.;Extraction of the cellular compounds by membrane rupture of the cells obtained in step c. or d.with a biomass / solvent ratio of between 1:5 and 1:50 (v / v) in an aqueous solvent with a pH of between 6 and 12 at a temperature of between 5°C and 45°C: Either by using ultrasound at a frequency of between 10 and 40 kHz, with a power of 50-1000 W for a duration of between 5 and 60 minutes; or Either by high pressure homogenization (HPH), from 1 to 4 cycles at a pressure of between 800 bars and 2500 bars; or Either by ball milling, with balls of diameter 0.4 to 2 mm, a filling of the extraction chamber of between 60 and 90%, at a rotation speed of 1000 to 4000 rpm for 15 to 90 minutes; Solid-liquid separation of the cells obtained in step e.by centrifugation at a speed of between 2,000 and 10,000 rpm for 5 to 30 min at a temperature of between 10°C and 20°C;Separation of the solid fraction comprising the proteins by precipitation with ammonium sulfate, with a saturation of 40% to 80%;Centrifugation of the precipitate obtained in step g. for 5 to 30 minutes, at a rotation speed of between 2,000 and 10,000 rpm at a temperature of 4 -15°C, making it possible to obtain a precipitate of hemin proteins;Recovery of the centrifugation pellet containing the protein precipitate and solubilization of the precipitate of hemin proteins obtained in step h. in an aqueous phase with a pH of between 6.5 and 12;Obtaining hemin proteins in aqueous solution.

[0066] Preferably, in order to obtain the hemin proteins in powder form, said method according to the invention further comprises: Drying the solution of hemin proteins obtained in step j by atomization in a flow of hot air at a temperature between 80°C and 300°C, preferably between 90°C and 220°C; Obtaining hemin proteins in powder form.

[0067] Microalgae are understood to mean all unicellular eukaryotic organisms (microalgae), as well as prokaryotic organisms such as cyanobacteria that can be cultivated heterotrophically. Among microalgae, the genera concerned are: Chlorella, Nannochloropsis, Scenedesmus, Tetraselmis, Phaeodactylum, Dunaliella, Isochrysis and Tisochrysis, preferably Nannochloropsis or Chlorella.

[0068] By heme protein (or hemoproteins) is meant: all heteroproteins containing a heme as a prosthetic group. These are metalloproteins for which the heme confers a functionality such as oxygen transport, oxygen reduction, electron transfer, etc. The heme, covalently or not bound to the protein, consists of an iron cation bound to the center of the conjugate base of the porphyrin.

[0069] Preferably, the iron-complexed heme proteins of interest are: cytochromes (cytochrome P450, cytochrome c oxidase, etc.) and certain enzymes such as ligninases, catalase, and peroxidases.

[0070] Cytochromes are intermediate oxidoreductases in the respiratory chain

[0071] Cytochromes P450 (CYP450) are hemoproteins (proteins with a heme molecule as a cofactor) that are involved in redox reactions

[0072] Cytochrome c oxidase is a membrane-bound oxidoreductase that contains two hemes and catalyzes the oxygen reduction reaction

[0073] Peroxidases are oxidase-type enzymes. In some cases, they are associated with a heme. Heme peroxidases include: Cyclooxygenase family: cyclooxygenase, Prostaglandin Endoperoxide H Synthases (PGHS), lactoperoxidases (LPO), myeloperoxidases (MPO), alpha-dioxygenase ('DiOx'). 2-heme peroxidases: cytochrome C peroxidases (DiHCcP), Catalase family: catalase DyP-type A / B / C / D peroxidase family Class I peroxidases: ascorbate peroxidases (APx) and cytochrome C peroxidases Class II peroxidases: lignin peroxidases

[0074] Ligninases are polymeric oxidoreductases that constitute a significant fraction of the plant cell wall. These enzymes include lignin peroxidase, manganese peroxidase, polyvalent peroxidase, and laccase-type phenol-oxidizing enzymes.

[0075] Catalase is a heme oxidoreductase formed from four polypeptide chains of approximately 500 amino acid residues, each containing a heme molecule.

[0076] Thus, preferably, the heme proteins within the meaning of the invention comprise at least one protein chosen from: cytochromes, cytochromes P450, cytochrome c oxidase, heme peroxidases, cyclooxygenase, prostaglandin endoperoxide H synthases, lactoperoxidases, myeloperoxidases, alpha-dioxygenases, cytochrome C peroxidases, catalase, DyP-type A / B / C / D peroxidases, ascorbate peroxidases, lignin peroxidase, manganese peroxidase, polyvalent peroxidase, and / or laccases.

[0077] According to a second aspect, the invention relates to a composition of hemin proteins as obtained by the method according to the invention.

[0078] This composition is in powder form or in aqueous solution form.

[0079] According to a third aspect, the invention also relates to the use of the hemin proteins obtained according to the invention for coloring a food, preferably a vegetable steak to imitate the characteristic browning of red meat during cooking.

[0080] Preferably, these heme proteins are included in a ready-to-use formulation for plant-based meat substitute applications.

[0081] According to a fourth aspect, the invention relates to a vegetable steak comprising the hemin protein composition obtained according to the invention as a colorant.

[0082] For the purposes of the invention, the term “vegetable steak” means a vegetable food product having the appearance of a red meat steak.

[0083] According to a fifth aspect, the invention relates to a meat substitute comprising the heme protein composition obtained according to the invention as a colorant.

[0084] By "meat substitute" is meant a product that replaces meat for food consumption.

[0085] According to the invention, the term "coloring" or "coloring agent" means an additive or foodstuff with coloring properties which makes it possible to give color to the product. In particular, within the framework of the invention, the coloring makes it possible to imitate the characteristic browning of red meat during cooking.

[0086] In particular, said hemin proteins according to the invention make it possible to imitate the characteristic browning of red meat during cooking.

[0087] According to a sixth aspect, the invention relates to the use of hemin proteins as a coloring agent for a food, making it possible to imitate the characteristic browning of red meat during cooking of said food.

[0088] Preferably, in said use, said hemin proteins are obtained by the method according to the invention.

[0089] Preferably, said food is a vegetable steak as a substitute for red meat steaks.

[0090] Preferably, said heme proteins comprise at least one protein chosen from: cytochromes, cytochromes P450, cytochrome c oxidase, heme peroxidases, cyclooxygenase, prostaglandin endoperoxide H synthases, lactoperoxidases, myeloperoxidases, alpha-dioxygenases, cytochrome C peroxidases, catalase, DyP-type A / B / C / D peroxidases, ascorbate peroxidases, lignin peroxidase, manganese peroxidase, polyvalent peroxidase, and / or laccases.

[0091] By "browning of red meat during cooking" is meant the change from a bright red color when the meat is raw to a darker red color, approaching brown / brown, when the outer layer of the meat is cooked.

[0092] By "imitate" is meant within the scope of the invention that the color of the cooked meat is reproduced.

[0093] In the description and in the following examples, unless otherwise indicated, the percentages are percentages by weight and the ranges of values ​​expressed in the form "between ... and ..." include the specified lower and upper limits. The examples below are presented for illustrative purposes and do not limit the scope of the invention. Examples

[0094] Example 1: Process for obtaining Nannochloropsis strains enriched in heme proteins by directed evolution in an oxidative stress environment.

[0095] Nannochloropsis was cultured by heterotrophy in a turbidostat medium supplemented with iron and subjected to oxidative stress (excess salt). Before the turbidostat culture step, the cells were cultured in 250 mL flasks with 150 mL of f / 2 culture medium for 4 days at 25°C with continuous shaking and a light source of 150 mol photons / m2 / s to acclimatize them.

[0096] The culture conditions shown in Table 1 were then used for directed evolution in turbidostat.

[0097] Culture parameter of example 1ParameterValuesInoculum10% (v / v) (30 mL in 300 mL)Temperature27 +-1 °CpH7.8 +-0.2Culture mediumCulture medium f / 2:Na NO3 (75.0 g / L dH2O)Na H2 PO4·H2O (5.0 g / L dH2O)Na2 SiO3 9H2O (30.0 g / L dH2O)Na2H2EDTA (4.36 g / L)FeCl3 6H2O (3.15 g / L)MnCl2 4H2O (180 mg / L)ZnSO4 (22 mg / L)CoCl2 6H2O (10 mg / L)CuSO4 5H2O (9.8 mg / L)Na2MoO4 2H2O (6.3 mg / L)Thiamine HCl (200 mg / L)Cyanocobalamin (10 mg / L)Biotin (100 mg / L)Filtered seawaterAutoclave the medium after adding all components.Iron supplementFeCl3 6H2O (5 g / L)Carbon source2 - 4 g / L glucoseStirring130 rpm continuouslyBrightnessNo lightSalt stress300 mM NaCl

[0098] Centrifuge the obtained microalgae cells for 15 min at a rotation speed of 1500 G and at a temperature of 15°C in order to remove the culture medium.

[0099] The protein-containing fraction is recovered and heme proteins are quantified by HPLC.

[0100] After quantification, contents are obtained which are 5% to 30% higher than in the case of a control strain cultivated under conventional conditions.

[0101] Example 2: Process for obtaining Chlorella enriched in heme proteins by directed evolution.

[0102] Chlorella was cultured by heterotrophy in a turbidostat in a medium supplemented with iron and subjected to oxidative stress (excess salt). Before the turbidostat culture step, the cells were cultured in 250 mL flasks with 150 mL of Knop culture medium for 4 days at 25°C with continuous shaking and with a light source of 150 mol photons / m 2 / s to acclimatize them.

[0103] The culture conditions shown in Table 2 were then used for directed evolution in turbidostat.

[0104] Culture parameter of example 2ParametersValuesInoculum10% (v / v)Temperature25 °CpH5.6 – 5.8CO25% (v / v)Culture mediumKnop's solution (g / L):1.25, KNO3; 1.25 KH2PO4; 2.5 MgSO47H2O; 0.30 Na citrate; 0.004 Fe2(SO4)3; 0.0029 H3BO3; 0.0018, MnCl24H2O; 0.00022 ZnSO47H2O; 0.000080 CuS045H2O; 0.000018 MoO3Carbon source10 g / L glucoseStirringContinuous, 150 rpmBrightnessNo lightSalt stress300 mM NaCl

[0105] Centrifuge the resulting algal cells for 12 min at a rotation speed of 2000 G and a temperature of 8°C to remove the culture medium.

[0106] The protein-containing fraction is recovered and heme proteins are quantified by HPLC.

[0107] After quantification by HPLC of the heme proteins obtained by directed evolution of Chlorella, contents are obtained which are 5 to 30% higher than in the case of a control, unselected strain.

[0108] Example 3: Extraction of Chlorella hemoproteins by high pressure homogenization (HPH)

[0109] Fresh chlorella biomass enriched in heme proteins by directed evolution is subjected to cell disruption by high-pressure homogenization according to the protocol in Table 3.

[0110] HPH parameter of example 3Extraction parametersValuesBiomass / solvent ratio (v / v)1:10Nature of solventDistilled waterpH8Pressure2000 barsNumber of cycles3Temperature35°C

[0111] After extraction, solid-liquid separation by centrifugation at a speed of 5,000 for 20 minutes at a temperature of 14°C is carried out in order to separate the cellular debris from the aqueous extract containing the heme proteins.

[0112] The protein fraction is then isolated by precipitation in a 60% saturated ammonium sulfate solution.

[0113] Finally, the precipitate is solubilized in an aqueous buffer at pH 7.

[0114] Example 4: Formulation of 430g of vegetable steak from the haem algal fraction.

[0115] 200 grams of black beans and 20 mL of water are blended in a blade blender at 500 rpm for 3 min.

[0116] A black bean puree is thus obtained.

[0117] The black bean puree is then mixed with 2g of garlic powder, 100g of chopped onion and 1g of heme protein according to the invention in powder form at a speed of 400 rpm for 5 min.

[0118] With continuous stirring at 400 rpm, 75 g of buckwheat flour, 30 g of corn starch, 1 g of salt and 1 g of pepper are gradually added.

[0119] This preparation is then left to stand for a quarter of an hour at room temperature.

[0120] Food products that visually resemble steaks are prepared by the user with their hands and then cooked for 3 minutes on each side in an oiled pan.

[0121] Thanks to the heme proteins according to the invention, the steak is red before cooking and turns brown after cooking.

Claims

A process for producing heme proteins in microalgae comprising the steps of: Acclimation of microalgae strains; Continuous culturing of said strains acclimated in step a. in a turbidostat under oxidative stress conditions for the cells of said strains, preferably by salt, light or metallic stress, optionally associated with an excess of iron; Harvesting of the microalgae cells obtained in step b. in order to remove the culture medium by flocculation, flotation, coagulation, filtration or centrifugation, preferably by centrifugation; Optionally, storage of the microalgae cells harvested in step c. at a temperature between -20°C and -90°C, preferably at -80°C, until extraction step e.; Extraction of cellular compounds by membrane rupture of the cells obtained in step c. or d.; ​​Solid-liquid separation of the cells obtained in step e., preferably by filtration, decantation or centrifugation, more preferably centrifugation; Separation of the solid fraction comprising the proteins obtained in step f., preferably by precipitation with ammonium sulfate, by size exclusion chromatography, by ion exchange chromatography, by filtration, , even more preferably by precipitation with ammonium sulfate; Centrifugation of the precipitate obtained in step g. allowing to obtain a precipitate of heme proteins; Recovery of the centrifuged pellet containing the precipitate of heme proteins and solubilization of the precipitate of heme proteins obtained in step h. in an aqueous phase; Obtaining heme proteins in aqueous solution. A process for producing heme proteins according to the preceding claim, wherein, in order to obtain the heme proteins in powder form, said process further comprises the steps of: Drying the heme protein solution obtained in step j, by lyophilization, spray drying, low-temperature drying or pressure drying, preferably by spray drying or lyophilization. Obtaining heme proteins in powder form. A process for producing heme proteins according to any one of the preceding claims, wherein in step b., said oxidative stress is selected from: Salt stress, comprising the use of a salt concentration of between 30 and 500 mM of NaCl for 1 to 5 days; or Light stress, by intermittent exposure to 30 and 500 µmol photons / m2 / s for 30 to 120 minutes, or prolonged exposure to a high light intensity of the order of 200 – 500 µmol photons / m2 / s for 0.5h to 72h; or Metal stress, by the addition of copper in concentrations of the order of 5 to 500 µM or concentrations of the order of 5 to 500 µg / L for 1 to 5 days. A process for producing heme proteins according to any one of the preceding claims, wherein step b. is carried out in association with an excess of iron in the culture medium, at a concentration of 0.5 to 40 mg / L. A process for producing heme proteins according to any one of the preceding claims, wherein step e is carried out by extracting cellular compounds by membrane rupture of the cells obtained in step c or d with a biomass / solvent ratio of between 1:5 and 1:50 (v / v) in an aqueous solvent with a pH of between 6 and 12 at a temperature of between 5°C and 45°C: By using ultrasound at a frequency of between 10 and 40 kHz, with a power of 50-1000 W for a duration of between 5 and 60 minutes; or By high-pressure homogenization, from 1 to 4 cycles at a pressure of between 800 bar and 2500 bar; or By ball milling, with balls of diameter 0.4 to 2 mm, filling the extraction chamber between 60 and 90%, at a rotation speed of 1000 to 4000 rpm for 15 to 90 minutes. A process for producing heme proteins according to any one of the preceding claims, wherein in step g. the separation of the solid fraction comprising the proteins by precipitation is carried out with ammonium sulfate, with a saturation of 40% to 80%. A process for producing heme proteins according to any one of the preceding claims, comprising the steps of: Acclimating microalgae strains, preferably in vials, at a temperature between 15°C and 35°C, preferably between 25°C and 30°C, for a period of between 3 and 6 days; Continuously culturing said strains acclimated in step a.in a turbidostat under oxidative stress conditions for the cells of said stems, said oxidative stress being chosen from: Salt stress, including the use of a salt concentration between 30 and 500 mM of NaCl for 1 to 5 days; or Light stress, by intermittent exposure to 30 and 500 µmol of photons / m2 / s for 30 to 120 minutes, or prolonged exposure to a high light intensity of the order of 200 – 500 µmol of photons / m2 / s for 0.5h to 72h; or Metallic stress, by adding copper in concentrations of the order of 5 to 500 µM or concentrations of the order of 5 to 500 µg / L for 1 to 5 days; said stress can optionally be associated with an excess of iron in the culture medium with a concentration of 0.5 to 40 mg / L. Harvesting of the microalgae cells obtained in step b.by centrifugation for 5 to 20 min at a rotation speed between 1000 and 2500 G and at a temperature between 10°C and 20°C, in order to remove the culture medium; Optionally, preservation of the microalgae cells harvested in step c. at a temperature between -20°C and -90°C, preferably at -80°C, pending step e. of extraction; Extraction of cellular compounds by membrane rupture of the cells obtained in step c. or d.with a biomass / solvent ratio between 1:5 and 1:50 (v / v) in an aqueous solvent with a pH between 6 and 12 at a temperature between 5°C and 45°C: By using ultrasound at a frequency between 10 and 40 kHz, with a power of 50-1000 W for a duration between 5 and 60 minutes; or By high-pressure homogenization, from 1 to 4 cycles at a pressure between 800 bars and 2500 bars; or By ball milling, with balls of diameter 0.4 to 2 mm, with the extraction chamber filled between 60 and 90%, at a rotation speed of 1000 to 4000 rpm for 15 to 90 minutes; Solid-liquid separation of the cells obtained in step e.by centrifugation at a speed between 2,000 and 10,000 rpm for 5 to 30 min at a temperature between 10°C and 20°C; Separation of the solid fraction including the proteins by precipitation with ammonium sulfate, with a saturation of 40% to 80%; Centrifugation of the precipitate obtained in step g. for 5 to 30 minutes, at a rotation speed between 2,000 and 10,000 rpm at a temperature of 4-15°C, allowing the obtaining of a heme protein precipitate; Recovery of the centrifuged pellet containing the heme protein precipitate and solubilization of the heme protein precipitate obtained in step h. in an aqueous phase with a pH between 6.5 and 12; Obtaining heme proteins in aqueous solution. A process for producing heme proteins according to the preceding claim, characterized in that, in order to obtain the heme proteins in powder form, said process further comprises the steps of: Drying the heme protein solution obtained in step j by atomization in a stream of hot air at a temperature between 80°C and 300°C, preferably between 90°C and 220°C. Obtaining heme proteins in powder form. A process for producing heme proteins according to any one of the preceding claims, wherein said microalgae are selected from: Chlorella, Nannochloropsis, Scenedesmus, Tetraselmis, Phaeodactylum, Dunaliella, Isochrysis and Tisochrysis, preferably Nannochloropsis or Chlorella. A process for producing heme proteins according to the preceding claim, wherein said microalgae are of the genus Nannochloropsis or Chlorella. A method for producing heme proteins according to any one of the preceding claims, wherein said heme proteins comprise at least one heme protein selected from: cytochromes, cytochromes P450, cytochrome c oxidase, heme peroxidases, cyclooxygenase, prostaglandin endoperoxide H synthases, lactoperoxidases, myeloperoxidases, alpha-dioxygenases, cytochrome C peroxidases, catalase, DyP-type A / B / C / D peroxidases, ascorbate peroxidases, lignin peroxidase, manganese peroxidase, polyvalent peroxidase, and / or laccases. Use of heme proteins as a food coloring agent, allowing to mimic the browning of red meat when cooking said food. Use according to the preceding claim, wherein said heme proteins are obtained by the process according to any one of claims 1 to 11. Use according to any one of claims 12 and 13, where said food is a plant-based steak as a substitute for red meat steaks. Use according to any one of claims 12 to 14, wherein said heme proteins comprise at least one protein selected from: cytochromes, cytochromes P450, cytochrome c oxidase, heme peroxidases, cyclooxygenase, prostaglandin endoperoxide H synthases, lactoperoxidases, myeloperoxidases, alpha-dioxygenases, cytochrome C peroxidases, catalase, DyP-type A / B / C / D peroxidases, ascorbate peroxidases, lignin peroxidase, manganese peroxidase, polyvalent peroxidase, and / or laccases.