EP3802782A1 - Method for cultivating unicellular red algae (ura) on a mixture of substrates - Google Patents

Method for cultivating unicellular red algae (ura) on a mixture of substrates

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Publication number
EP3802782A1
EP3802782A1 EP19725747.0A EP19725747A EP3802782A1 EP 3802782 A1 EP3802782 A1 EP 3802782A1 EP 19725747 A EP19725747 A EP 19725747A EP 3802782 A1 EP3802782 A1 EP 3802782A1
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EP
European Patent Office
Prior art keywords
biomass
glucose
glycerol
culture
carbon source
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19725747.0A
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German (de)
French (fr)
Inventor
Olivier CAGNAC
Axel ATHANE
Marion CHAMPEAUD
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Fermentalg SA
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Fermentalg SA
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Publication date
Application filed by Fermentalg SA filed Critical Fermentalg SA
Publication of EP3802782A1 publication Critical patent/EP3802782A1/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G33/00—Cultivation of seaweed or algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/40—Colouring or decolouring of foods
    • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
    • A23L5/46—Addition of dyes or pigments, e.g. in combination with optical brighteners using dyes or pigments of microbial or algal origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management

Definitions

  • the present invention relates to the cultivation of ARU for the production of biomass for the production of products of interest, such as dried biomass or compounds or mixtures of compounds of interest extracted from the biomass produced, in particular pigments or food coloring.
  • the invention relates more particularly to the industrial production of this biomass, which must respond to an economic balance of profitability, with on the one hand an increase in the productivity (quantity of biomass and of compounds of interest in the biomass) and on the one hand other an economically acceptable production cost.
  • Phycocyanins are pigments produced by unicellular microorganisms that can be used for food coloring. Today, they are mainly produced by the culture of cyanobacteria, basins and autotrophy, low yield of biomass produced. To increase the production of these phycocyanins, phycocyanin producing species have been sought and capable of being cultured under bioreactor fermentation conditions in order to increase the amount of biomass produced.
  • Unicellular red algae (ARU) belonging to the class of cyanidiophyceae such as Galdieria sulphuraria are known to produce phycocyanins (Carfagna & al., 2018;) capable of developing under autotrophic, mixotrophic and heterotrophic conditions.
  • the addition of one or more carbon sources in the culture medium makes it possible to significantly increase the growth rate.
  • These microalgae are capable of consuming a large number of carbonaceous metabolites, in total more than one groin has been counted (glucose, glycerol, pentose, etc.) (Gross et al., 1995, Oesterhelt et al., 1999). - 2007, Sloth & al, 2006).
  • the present invention relates to a new method for the cultivation of unicellular red algae (ARU) for the production of a biomass rich in phycocyanins, comprising the steps of (i) mixing in the mixotrophic or heterotrophic mode of said ARUs on a culture medium comprising a carbon source comprising glucose, and (ii) biomass recovery, characterized in that there is added to the culture medium an amount of glycerol sufficient to increase the production of phycocyanin relative to the culture without glycerol.
  • ARU unicellular red algae
  • the invention also relates to a process for preparing phycocyanins which comprises producing a biomass according to the invention and a step (iii) for extracting phycocyanins from the previously recovered biomass.
  • Figure 1 shows the growth monitoring curves and production of phycocyanins under the culture conditions of Example 1.
  • the present invention relates to a new method for the cultivation of unicellular red algae (ARU) for the production of a biomass rich in phycocyanins, comprising the steps of (i) mixing in the mixotrophic or heterotrophic mode of said ARUs on a culture medium comprising a carbon source comprising glucose, and (ii) biomass recovery, characterized in that there is added to the culture medium an amount of glycerol sufficient to increase the production of phycocyanin relative to the culture without glycerol.
  • ARU unicellular red algae
  • ARUs used to produce biomass by fermentation and fermentation methods, particularly for the production of phycocyanins are well known to those skilled in the art.
  • patent applications WO 2017/050917, WO 2017/093345, WO 2017/050918 and FR 1752674 filed on March 30, 2017 are cited in particular.
  • the ARUs are in particular chosen from the sub-division of Cyanidiophytina, in particular from the class of Cyanidiophyceae, more particularly from the order of cyanidiales, even more particularly chosen from the families of Cyanidiaceae or Galdieriaceae.
  • the ARUs are chosen from the genera Cyanidioschyzon, Cyanidium or Galdieria.
  • the ARUs employed in the process according to the invention are chosen from the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or still Galdieria sulphuraria.
  • These culture media comprise a carbon source comprising glucose. It may be glucose or glucose in a complex form such as lactose, fructose or polysaccharides comprising glucose.
  • This source of carbon can come from the sugar industry, beet or cane, starch hydrolysates from starch plants such as corn, wheat, potatoes, or from the dairy industry such as milk permeate. (WO 2017/093345), high in lactose, used alone or in a mixture.
  • the carbon source comprising glucose is selected from glucose and lactose.
  • the culture medium generally comprises the carbon source comprising glucose in an amount of between 0.05 g / l and 200 g / l, advantageously between 1 g / l and 150 g / l, very advantageously between 10 g / l and 80 g / l. g / L.
  • the culture medium may comprise other elements well known to those skilled in the field of fermentation microalgae culture, in particular a source of phosphorus and / or a source of nitrogen, and / or a source of sulfur.
  • the sources of phosphorus can be chosen from among the following species: phosphoric acid, phosphorus salts, advantageously sodium hydrogenphosphorus (Na2HPC> 4), or sodium dihydrogenophosphorus (NahhPCU), or potassium dihydrogénophosphore (KH2PO4), or potassium hydrogénophosphore (K 2 HPO 4), or mixture, in any proportion of two or more of these sources.
  • the medium may also include macroelements and microelements that promote the culture of ARU.
  • the lighting conditions for the mixotrophic culture of the ARUs are also known to a person skilled in the art, in particular described in the patent applications WO 2017/050917, WO 2017/050918, WO 2017/093345 and FR 1752674 filed on 30 March 2017.
  • Lighting may be continuous or discontinuous, in particular discontinuous in the form of flashes.
  • the illumination is carried out with a blue light (WO 2017/050917), more particularly in the form of a radiation having a narrow spectrum of wavelength between 400 and 550 nm, advantageously a narrow spectrum wavelength between 420 nm and 500 nm, preferably between 430 and 480 nm, very preferably centered on 455 nm.
  • a blue light WO 2017/050917
  • the invention is characterized in that glycerol is added to the culture medium in an amount sufficient to increase the production of phycocyanins relative to the culture without glycerol.
  • This addition can be made in the culture medium at the beginning of culture, or in culture media, once the biomass has reached a specific density, for example greater than 20 g / l of dry matter in the culture medium.
  • the step (i) of culture can therefore be divided into two substeps, (ia) of growth to produce biomass with a carbon source comprising essentially glucose in a simple or complex form as defined above and (ib) ) of accumulation with the addition of glycerol to promote the production of phycocyanin.
  • Industrial glycerol sources that can be integrated into production processes have no restriction on purity or refining (generally between 80% and 100%). Preferably they are food grade. Glycerol source suppliers are well known, particularly players in the biodiesel industry such as the Avril group with Oléon (Glycerine 4808 / 4808K, Glycerin 4827 / 4827K ...), or Cargill, which also offers a wide range of products. a product range based on glycerine refined between 86.5% and 99.7% possessing Kosher, Halal, RSPO, E442 or GMO-free certifications, among others.
  • Glycerol source suppliers are well known, particularly players in the biodiesel industry such as the Avril group with Oléon (Glycerine 4808 / 4808K, Glycerin 4827 / 4827K ...), or Cargill, which also offers a wide range of products. a product range based on glycerine refined between 86.
  • the carbon source comprising glucose is the main source of carbon in the culture medium and the amount of glycerol sufficient to increase the production of phycocyanin relative to the culture without glycerol will be readily determined by those skilled in the art by a simple experiment. fermenters for comparing the amount of phycocyanin produced with or without glycerol as shown in the examples.
  • the desired increase in phycocyanin production will preferably be at least 0.15 mg / g / h, more preferably at least 0.30 mg / g / h, more preferably at least 1 mg / g / h; or at least 3.6 mg / g, more preferably at least 7.2 mg / g, more preferably at least 24 mg / gX.
  • the amount of glycerol added to the culture medium is sufficient to have a glycerol / carbon source weight ratio comprising glucose (hereinafter referred to as Gly / Glu) of at least 1/15.
  • Gly / Glu glycerol / carbon source weight ratio comprising glucose
  • the Gly / Glu ratio is at least 1/14, more preferably at least 1/10, even more preferably at least
  • the interest is to ensure optimum profitability between the cost of raw materials and the production of phycocyanin, it will be advantageous not to exceed a Gly / Glu ratio of 1/1.
  • the glycerol is added to the culture medium for a Gly / Glu weight ratio of 1/10 to 1/1, preferably 1/7 to 1/2, more preferably about 1/5 to about 1/3.
  • the method according to the invention may further comprise a step of recovering the biomass.
  • Said recovery of the biomass can be carried out by any technique allowing the recovery of biomass, including filtration methods, gravimetric or under reduced pressure, decantation, or else precipitation methods followed by gravimetric filtration.
  • the invention also relates to the biomass obtainable by any of the variants of the process according to the invention.
  • biomass according to the invention is advantageously understood to mean a set of microorganism cells produced by the culture thereof, which cells may or may not have retained their physical integrity. It is thus understood that said biomass may comprise an amount of degraded microorganism cells ranging from 0% to 100%. By “degraded” it is meant that the physical integrity of said microorganism cells may have been altered such as, for example, lysed microorganisms, resulting for example from a homogenization or enzymatic lysis process. Once produced, this biomass can be raw, just separated from its culture medium, dried or not, degraded or not.
  • Biomass depending on whether it is dried or not, in whole or in part, may comprise a moisture content of 1% to 90%.
  • the biomass has a moisture content of 70% to 90%, preferably 80% to 85%. This is particularly the case when it consists essentially of industrial microorganisms, optimized and cultured, after filtration of the fermentation must to separate the cultured microorganisms from the culture medium, before drying.
  • the biomass is dried in whole or in part and has a moisture content of 1% to 10%, preferably 2% to 7%.
  • said biomass may have a density in ARUs of between 20 and 200 g / l of dry matter, preferably between 90 and 150 g / l of dry matter.
  • said biomass may have a protein content of between 25% and 60%, even up to 70%, preferably between 30% and 55%, more preferably between 40% and 50% by weight of dry matter.
  • the determination of nitrogen and the crude protein content is calculated using the bulk digestion method and steam distillation (NF EN ISO 5983-2).
  • said biomass may have an intracellular content of phycobiliproteins (phycocyanin and allophycocyanin) of between 1 and 250 mg / g of dry matter, preferably between 20 and 150 mg / g of dry matter.
  • phycobiliproteins phycocyanin and allophycocyanin
  • said biomass may have an intracellular phycocyanin content of between 0.5 and 100 mg / g of dry matter, preferably between 10 and 40 mg / g of dry matter.
  • Biomass may be conditioned for storage or for its use as such, for example as a food supplement or food for human or animal consumption.
  • the cake that can be obtained after extraction of the phycocyanin from the biomass of ARUs that can be obtained by the method according to the invention can be used as a dietary supplement rich in proteins and carotenoids, in human food or animal.
  • said biomass may have an intracellular content of carotenoids of between 0.1 and 10 mg / g of dry matter, advantageously between 0.250 and 1 mg / g of dry matter.
  • ARUs have a significant potential for use in many fields, such as, for example, human or animal nutrition, cosmetics and medicine.
  • said biomass of ARUs obtainable according to the invention can be used after harvest either directly, optionally dried or after transformation.
  • said biomass may be used in the form of flours entering food compositions or in the form of food supplements.
  • the biomass of ARUs that can be obtained according to the invention can be transformed into flour according to any method known to those skilled in the art. It can thus be envisaged, for example, that the ARUs can be separated from the culture medium, lysed and reduced to fine particles (average diameter of 10 microns), and then dried.
  • the invention also relates to any use of the biomass of ARUs obtainable according to the invention in any known field of use of ARUs, particularly, food or feed, cosmetics, medicine. In the fields of human or animal nutrition and cosmetics, these are of course non-therapeutic uses for animals or healthy humans.
  • the biomass obtained after culture of ARUs according to the method of the invention can make it possible to obtain in particular a flour rich in antioxidants, in particular carotenoids (particularly zeaxanthin and b-carotenes) in contents of between 0.1 and 10 mg / g of dry matter, advantageously between 0.25 and 1 mg / g of dry matter, in particular zeaxanthin in a content of between 0 and 10 mg / g.
  • a flour rich in antioxidants in particular carotenoids (particularly zeaxanthin and b-carotenes) in contents of between 0.1 and 10 mg / g of dry matter, advantageously between 0.25 and 1 mg / g of dry matter, in particular zeaxanthin in a content of between 0 and 10 mg / g.
  • 05 and 5 mg / g of dry matter advantageously between 0.1 and 1 mg / g of dry matter, and / or of b-carotene at a content of 0.05 and 5 mg / g of dry matter, advantageously between 0 and , 1 and 1 mg / g of dry matter, meeting a need particularly in the food industry, because more palatable, having better taste, providing antioxidants in large quantities and can be used in animal or human food.
  • the invention therefore relates to a flour that can be obtained after transformation of the biomass into ARUs that can be obtained by the process according to the invention.
  • said product can be used pure or mixed with other conventionally used ingredients, particularly in non-organic uses. therapeutics in food or cosmetics.
  • the invention also relates to any product that may comprise at least the algal biomass obtainable according to the invention.
  • the invention also relates to any product which may comprise at least flour resulting from the transformation of the algal biomass obtainable according to the invention.
  • the phycocyanins produced by said biomass can be extracted for use, for example, in the diet or as a dye.
  • the extraction of phycobiliproteins, and particularly phycocyanin, from said biomass may be carried out according to any extraction technique known to those skilled in the art such as that described by Moon et al. (2014) or by Jaouen & al. (1999) or in the applications FR 2 789 399, WO 2017/093345 and FR 1752674 filed March 30, 2017.
  • the invention also relates to the use of phycocyanin obtainable according to the method of the invention, in food, animal or human, as a dietary supplement, or as a dye, particularly as food coloring.
  • Example 1 Comparative growth of the G. sulphuraria strain on glycerol, glucose and a glucose-glycerol mixture.
  • the growth of the strain is carried out by measuring the absorbance at 800 nm over time.
  • the culture medium is the one known for this strain (medium Gross), the the only difference between the three media tested is the carbon substrate (s) used.
  • Culture medium 30g / L of glucose or 30g / L of glycerol (glycerin 4808 Univar) or 25g / L of glucose + 5g / L of glycerol; 8 g / L of (NH4) 2SO4; 0.25 g / L of KH 2 PO 4; 716 mg / L MgSO4; 44mg / L CaCl2; 3 ml / L of Fe-EDTA stock solution (FeS04 at 6.9 g / L and Na2-EDTA at 9.3 g / L) and 4 ml of metal trace stock solution (3.09 g / L of Na2-EDTA; 08g / L CuS04.5H2O, 2.86 g / L H3B03, 0.04g / L NaV03.4H2O, 1.82g / L MnCl2, 0.04g / L CoCl2.6H2O, 0.22g / L ZnSO4.7H2O, 0.017g / L Na2
  • Culture conditions The cultures are carried out on a stirring table (140 rpm) in a thermostatically controlled enclosure at 37 ° C. in the middle as described above in the presence of a light source.
  • Crop monitoring The growth monitoring shows that we achieve similar OD regardless of the source (s) of carbon (s) used, dry masses and associated phycocyanin assays after 312h of culture are described In the picture.
  • glycerol in the medium activates the production of phycocyanin despite the presence of glucose known as phycocyanin inhibitor.
  • the growth of the strain is carried out by measuring the absorbance at 800 nm over time.
  • the culture medium is that known for this strain (medium Gross), the only difference between the two media tested is the carbon substrate (s) used (s).
  • Culture medium 30g / L of milk permeate or 25g / L of milk permeate + 5g / L of glycerol (glycerin 4808 Univar); 8 g / L of (NH4) 2SO4; 0.25 g / L of KH 2 PO 4; 716 mg / L MgSO4; 44mg / L CaCl2; 3 ml / L of Fe-EDTA stock solution (FeS04 at 6.9g / L and Na2-EDTA at 9.3 g / L) and 4 ml of metal trace stock solution (3.09 g / L Na2-EDTA; 08g / L CuS04.5H2O, 2.86g / L H3B03, 0.04g / L NaV03.4H2O, 1.82g / L MnCl2, 0.04g / L CoCl2.6H2O; 0.22g / L ZnSO4.7H2O; 0, 017g / L Na2SeO3; 0.03 g
  • Culture conditions The cultures are carried out on a stirring table (140 rpm) in a thermostatically controlled enclosure at 37 ° C., in the medium as described above, in the presence or absence of a light source.
  • Crop Monitoring The dry masses and associated phycocyanin assays after 168 h of culture are described in Table 2 (A) in the presence of light and in Table 2 (B) in the dark.
  • Example 3 follow-up of a bioreactor culture method of the G.sulphuraria strain on a glucose-glycerol mixture.
  • the growth of the strain is monitored by measuring the dry mass over time and the substrate consumption is identified by HPLC assay. An intracellular phycocyanin assay is also performed over time.
  • the culture medium is that known for this strain (medium Gross) with 1/6 substrate of glycerol and 5/6 of glucose for the stock and a diet consisting of 1 ⁇ 4 glycerol and 3 ⁇ 4 glucose for the fed-batch .
  • Foot of tank 25g / L glucose + 5g / L of glycerol (glycerin 4808 Univar); 8g / L of
  • Culture conditions The cultures are carried out in a 2L bioreactor at a temperature controlled at 37 ° C., pH 3, in the presence of a light source.
  • Example 4 follow-up of a bioreactor culture method of the G.sulphuraria strain on a permeate mixture of milk-glycerol.
  • the growth of the strain is monitored by measuring the dry mass over time and the substrate consumption is identified by HPLC assay. An intracellular phycocyanin assay is also performed over time.
  • the culture medium is the one known for this strain (medium Gross) with for 1/6 substrate of glycerol and 5/6 of lactose (brought by the permeate of milk) for the base of vat and a feed composed of 1 ⁇ 4 glycerol and 3 ⁇ 4 of lactose for fed-batch.
  • Foot of tank 25g / L permeate of milk + 5g / L of glycerol (Glycerin 4808 Univar); 8 g / L of (NH4) 2SO4; 716 mg / L MgSO4; 3 ml / L of Fe-EDTA stock solution (FeS04 at 6.9 g / L and Na2-EDTA at 9.3 g / L) and 4 ml of metal trace stock solution (3.09 g / L of Na2-EDTA; 08g / L CuS04.5H2O, 2.86 g / L H3B03, 0.04g / L NaV03.4H2O, 1.82g / L MnCl2, 0.04g / L CoCl2.6H2O, 0.22g / L ZnSO4.7H2O, 0.017g / L Na2SeO3 0.03g / L
  • Culture conditions The cultures are carried out in a 2L bioreactor at a temperature controlled at 37 ° C., pH 3, in the presence of a light source.
  • Example 5 Variation of PC Production by Gly / GIc Proportion in the culture medium.
  • a cross-over range of glucose and glycerol was performed to evaluate the effect of glycerol on the inhibition of inhibition of phycocyanin synthesis related to the presence of glucose in the culture medium.
  • the total amount of organic carbon in each Erlenmeyer flask is 30 g / l.
  • Glucose is symbolized by the contraction "glc” and glycerol by "gly”.
  • the concentration of each element in the medium is shown in Table 3.
  • Example 7 Variation of PC Production by Glycerol Proportion brought into a medium containing at the beginning of the culture 30g / l of milk permeate.
  • glycerol Various amounts of glycerol are added to a medium composed of milk permeate in order to observe the effect of this on the phycocyanin synthesis and the removal of the lactose (glucose + sucrose) inhibition on the production of this pigment .
  • the base medium contains 30g / l of milk permeate.
  • the added glycerol is symbolized by the contraction "gly”. 6 glycerol concentrations are tested (0, 1, 2, 3, 4 and 5g / l).
  • glucose has a limiting effect on the amount of phycocyanin produced by the Galdieria sulphuraria strain, with a concentration of less than 15 mg of phycocyanin per g of dry matter at the end of growth (15 mg PC / g of MS).
  • the strain having grown in the presence of glycerol only accumulates about 40 mg PC / g of MS, for the same culture time (240h).
  • the culture grown in glycerol may have accumulated up to 70 mg of PC / g of MS, while the one having grown in glucose will have a quantity of PC / g of MS which will remain more or less constant over time.
  • the addition of small amounts of glycerol in the medium makes it possible to eliminate the inhibition of PC synthesis linked to the presence of glucose in the medium, whether it is present in the form of simple sugar (glucose), or complex (lactose, sucrose, or other ).
  • This method makes it possible to grow the cells using glucose (or a carbon source containing glucose) as the main carbon source, and to use glycerol only as a PC synthesis inducer and not as a main substrate.
  • Glucose or carbon sources containing glucose are usually cheaper than glycerol, and generally allow to obtain growth rates higher than glycerol, thus saving on fermentation costs.

Abstract

The invention relates to the cultivation of URA for producing biomass for the production of products of interest, such as dry biomass or compounds or mixtures of compounds of interest extracted from the biomass produced, particularly food pigments or colouring agents. The invention more particularly relates to the industrial production of said biomass, which must satisfy an economic equilibrium of profitability, with both an increase in productivity (quantity of biomass and of compounds of interest in the biomass) and an economically acceptable production cost.

Description

PROCÉDÉ DE CULTURE D’ALGUES ROUGES UNICELLULAIRES (ARU) SUR UN  PROCESS FOR CULTIVATION OF UNICELLULAR RED ALGAE (ARU) ON A
MÉLANGE DE SUBSTRATS  MIXING SUBSTRATES
DOMAINE DE L'INVENTION FIELD OF THE INVENTION
La présente invention concerne la culture d’ARU pour la production de biomasse en vue de la production de produits d’intérêts, comme la biomasse séchée ou des composés ou mélanges de composés d’intérêt extraits de la biomasse produite, en particulier des pigments ou colorants alimentaires. L’invention concerne plus particulièrement la production industrielle de cette biomasse, qui doit répondre à un équilibre économique de rentabilité, avec d’un côté une augmentation de la productivité (quantité de biomasse et de composés d’intérêt dans la biomasse) et de l’autre un coût de production économiquement acceptable.  The present invention relates to the cultivation of ARU for the production of biomass for the production of products of interest, such as dried biomass or compounds or mixtures of compounds of interest extracted from the biomass produced, in particular pigments or food coloring. The invention relates more particularly to the industrial production of this biomass, which must respond to an economic balance of profitability, with on the one hand an increase in the productivity (quantity of biomass and of compounds of interest in the biomass) and on the one hand other an economically acceptable production cost.
ETAT DE LA TECHNIQUE STATE OF THE ART
Les phycocyanines sont des pigments produits par des microorganismes unicellulaires qui peuvent être employés pour la coloration des aliments. Aujourd’hui, elles sont essentiellement produites par la culture de cyanobactéries, en bassins et en autotrophie, à faible rendement en biomasse produite. Pour augmenter la production de ces phycocyanines, on a cherché des espèces productrices de phycocyanines et capables d’être cultivées dans des conditions de fermentation en bioréacteur afin d’augmenter la quantité de biomasse produite.  Phycocyanins are pigments produced by unicellular microorganisms that can be used for food coloring. Today, they are mainly produced by the culture of cyanobacteria, basins and autotrophy, low yield of biomass produced. To increase the production of these phycocyanins, phycocyanin producing species have been sought and capable of being cultured under bioreactor fermentation conditions in order to increase the amount of biomass produced.
Les algues rouges unicellulaires (ARU) appartenant à la classe des cyanidiophyceae comme Galdieria sulphuraria sont connues pour produire des phycocyanines (Carfagna & al., 2018 ; ) capables de se développer en conditions autotrophe, mixotrophe et hétérotrophe. L’ajout d’une ou plusieurs sources carbonées dans le milieu de culture permet d’augmenter significativement la vitesse de croissance. Ces micro-algues sont capables de consommer un nombre important de métabolites carbonés, au total plus d’une 30aine ont été dénombrés (glucose, glycérol, pentose...) (Gross et al., 1995 ; Oesterhelt et al., 1999 - 2007 ; Sloth & al, 2006). Cependant, malgré une croissance plus rapide, le contenu cellulaire en phycocyanines est bien moindre en conditions hétérotrophes et mixotrophes qu’en autotrophie. L’ajout d’une source carbonée dans le milieu de culture - en présence ou non de lumière - réprime fortement la production de phycocyanines (Stadnichuk et al., 1998). Unicellular red algae (ARU) belonging to the class of cyanidiophyceae such as Galdieria sulphuraria are known to produce phycocyanins (Carfagna & al., 2018;) capable of developing under autotrophic, mixotrophic and heterotrophic conditions. The addition of one or more carbon sources in the culture medium makes it possible to significantly increase the growth rate. These microalgae are capable of consuming a large number of carbonaceous metabolites, in total more than one groin has been counted (glucose, glycerol, pentose, etc.) (Gross et al., 1995, Oesterhelt et al., 1999). - 2007, Sloth & al, 2006). However, despite faster growth, the cellular content of phycocyanins is much lower in heterotrophic and mixotrophic conditions than in autotrophy. The addition of a carbon source in the culture medium - with or without light - strongly suppresses phycocyanin production (Stadnichuk et al., 1998).
Pour compenser cette baisse de production intrinsèque de phycocyanine, des méthodes de culture ont été développées pour augmenter la densité cellulaire dans les fermenteurs et la quantité de matière sèche produite. En augmentant la matière sèche, on augmente également la quantité de phycocyanine produite (WO 2017/050917, WO 2017/093345). On peut également, selon les ARU employées, produire des phycocyanines aux propriétés nouvelles, comme celle d’avoir une meilleure stabilité à pH acide (WO 2017/050918, FR 3 064 365 & WO 2018/178334). To compensate for this decline in intrinsic phycocyanin production, culture methods have been developed to increase the cell density in fermentors and the amount of dry matter produced. By increasing the dry matter, the amount of phycocyanin produced is also increased (WO 2017/050917, WO 2017/093345). Depending on the ARU used, it is also possible to produce phycocyanines with novel properties, such as having a better stability at acidic pH (WO 2017/050918, FR 3,064,365 & WO 2018/178334).
Une amélioration de ces procédés de production permettrait de combiner à la fois les avantages d’une production industrielle par fermentation sur substrat carboné, tout en diminuant la répression de la production des phycocyanines associée à la source de carbone.  An improvement in these production processes would make it possible to combine the advantages of an industrial production by fermentation on a carbon substrate, while decreasing the repression of the production of phycocyanins associated with the carbon source.
EXPOSE DE L'INVENTION SUMMARY OF THE INVENTION
La présente invention concerne un nouveau procédé de culture d’algues rouges unicellulaires (ARU) pour la production d’une biomasse riche en phycocyanines, comprenant les étapes (i) de cultures en mode mixotrophe ou hétérotrophe desdites ARU sur un milieu de culture comprenant une source de carbone comprenant du glucose, et (ii) de récupération de la biomasse, caractérisé en ce que l’on ajoute au milieu de culture une quantité de glycérol suffisante pour augmenter la production de phycocyanine par rapport à la culture sans glycérol.  The present invention relates to a new method for the cultivation of unicellular red algae (ARU) for the production of a biomass rich in phycocyanins, comprising the steps of (i) mixing in the mixotrophic or heterotrophic mode of said ARUs on a culture medium comprising a carbon source comprising glucose, and (ii) biomass recovery, characterized in that there is added to the culture medium an amount of glycerol sufficient to increase the production of phycocyanin relative to the culture without glycerol.
L’invention concerne aussi un procédé de préparation de phycocyanines qui comprend la production d’une biomasse selon l’invention et une étape (iii) d’extraction des phycocyanines à partir de la biomasse préalablement récupérée.  The invention also relates to a process for preparing phycocyanins which comprises producing a biomass according to the invention and a step (iii) for extracting phycocyanins from the previously recovered biomass.
DESCRIPTION DES FIGURES DESCRIPTION OF THE FIGURES
La Figure 1 représente les courbes de suivi de croissance et de production de phycocyanines dans les conditions de culture de l’exemple 1.  Figure 1 shows the growth monitoring curves and production of phycocyanins under the culture conditions of Example 1.
DESCRIPTION DETAILLEE DE L'INVENTION DETAILED DESCRIPTION OF THE INVENTION
La présente invention concerne un nouveau procédé de culture d’algues rouges unicellulaires (ARU) pour la production d’une biomasse riche en phycocyanines, comprenant les étapes (i) de cultures en mode mixotrophe ou hétérotrophe desdites ARU sur un milieu de culture comprenant une source de carbone comprenant du glucose, et (ii) de récupération de la biomasse, caractérisé en ce que l’on ajoute au milieu de culture une quantité de glycérol suffisante pour augmenter la production de phycocyanine par rapport à la culture sans glycérol.  The present invention relates to a new method for the cultivation of unicellular red algae (ARU) for the production of a biomass rich in phycocyanins, comprising the steps of (i) mixing in the mixotrophic or heterotrophic mode of said ARUs on a culture medium comprising a carbon source comprising glucose, and (ii) biomass recovery, characterized in that there is added to the culture medium an amount of glycerol sufficient to increase the production of phycocyanin relative to the culture without glycerol.
Les ARU employées pour produire de la biomasse par fermentation et les méthodes de fermentation, en particulier pour la production de phycocyanines sont bien connues de l’homme du métier. On citera en particulier les demandes de brevet WO 2017/050917, WO 2017/093345, WO 2017/050918 et FR 1752674 déposée le 30 mars 2017.  ARUs used to produce biomass by fermentation and fermentation methods, particularly for the production of phycocyanins are well known to those skilled in the art. In particular, patent applications WO 2017/050917, WO 2017/093345, WO 2017/050918 and FR 1752674 filed on March 30, 2017 are cited in particular.
Les ARU sont en particulier choisies parmi la sous-division des Cyanidiophytina, en particulier de la classe des Cyanidiophyceae, plus particulièrement de l'ordre des Cyanidiales, encore plus particulièrement choisies parmi les familles des Cyanidiaceae ou des Galdieriaceae. De préférence, les ARU sont choisies parmi les genres Cyanidioschyzon, Cyanidium ou Galdieria. Plus préférentiellement, les ARU employées dans le procédé selon l’invention sont choisies parmi les espèces Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201 , Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita ou encore Galdieria sulphuraria. The ARUs are in particular chosen from the sub-division of Cyanidiophytina, in particular from the class of Cyanidiophyceae, more particularly from the order of cyanidiales, even more particularly chosen from the families of Cyanidiaceae or Galdieriaceae. Preferably, the ARUs are chosen from the genera Cyanidioschyzon, Cyanidium or Galdieria. More preferably, the ARUs employed in the process according to the invention are chosen from the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or still Galdieria sulphuraria.
Les milieux de culture employés dans le procédé selon l’invention sont bien connus de l’homme du métier, en particulier décrits dans les demandes de brevet WO 2017/050917, WO 2017/093345, WO 2017/050918 et FR 1752674 déposée le 30 mars 2017.  The culture media used in the process according to the invention are well known to those skilled in the art, in particular described in patent applications WO 2017/050917, WO 2017/093345, WO 2017/050918 and FR 1752674 filed on 30 March 2017.
Ces milieux de culture comprennent une source de carbone comprenant du glucose. Il peut s’agir de glucose ou encore de glucose sous une forme complexe comme le lactose, le fructose ou des polyosides comprenant du glucose.  These culture media comprise a carbon source comprising glucose. It may be glucose or glucose in a complex form such as lactose, fructose or polysaccharides comprising glucose.
Cette source de carbone peut provenir de l’industrie sucrière, de betterave ou de canne, des hydrolysats d’amidons de plantes amidonnées comme le maïs, le blé, la pomme de terre, ou encore de l’industrie laitière comme le perméat de lait (WO 2017/093345), riche en lactose, employés seuls ou en mélange.  This source of carbon can come from the sugar industry, beet or cane, starch hydrolysates from starch plants such as corn, wheat, potatoes, or from the dairy industry such as milk permeate. (WO 2017/093345), high in lactose, used alone or in a mixture.
De manière avantageuse, la source de carbone comprenant du glucose est choisie parmi le glucose et le lactose.  Advantageously, the carbon source comprising glucose is selected from glucose and lactose.
Le milieu de culture comprend généralement la source de carbone comprenant du glucose en une quantité comprise entre 0,05 g/L et 200 g/L, avantageusement entre 1 g/L et 150g/L, très avantageusement entre 10 g/L et 80 g/L.  The culture medium generally comprises the carbon source comprising glucose in an amount of between 0.05 g / l and 200 g / l, advantageously between 1 g / l and 150 g / l, very advantageously between 10 g / l and 80 g / l. g / L.
Le milieu de culture peut comprendre d’autres éléments bien connus de l’homme du métier dans le domaine de la culture des microalgues par fermentation, en particulier une source de phosphore et/ou une source d’azote, et/ou une source de soufre.  The culture medium may comprise other elements well known to those skilled in the field of fermentation microalgae culture, in particular a source of phosphorus and / or a source of nitrogen, and / or a source of sulfur.
Selon l'invention, les sources de phosphore peuvent être choisies parmi les espèces suivantes : l’acide phosphorique, les sels de phosphore, avantageusement de l’hydrogénophosphore de sodium (Na2HPC>4), ou du dihydrogénophosphore de sodium (NahhPCU), ou du dihydrogénophosphore de potassium (KH2PO4), ou de l’hydrogénophosphore de potassium (K2HPO4), ou tout mélange, en toute proportion d'au moins deux de ces sources. According to the invention, the sources of phosphorus can be chosen from among the following species: phosphoric acid, phosphorus salts, advantageously sodium hydrogenphosphorus (Na2HPC> 4), or sodium dihydrogenophosphorus (NahhPCU), or potassium dihydrogénophosphore (KH2PO4), or potassium hydrogénophosphore (K 2 HPO 4), or mixture, in any proportion of two or more of these sources.
Le milieu peut aussi comprendre des macroéléments et des microéléments qui favorisent la culture de ARU.  The medium may also include macroelements and microelements that promote the culture of ARU.
Les conditions d’éclairage pour la culture en mode mixotrophe des ARU sont également connues de l’homme du métier, notamment décrites dans les demandes de brevet WO 2017/050917, WO 2017/050918, WO 2017/093345 et FR 1752674 déposée le 30 mars 2017.  The lighting conditions for the mixotrophic culture of the ARUs are also known to a person skilled in the art, in particular described in the patent applications WO 2017/050917, WO 2017/050918, WO 2017/093345 and FR 1752674 filed on 30 March 2017.
L’éclairage peut être continu ou discontinu, notamment discontinu sous la forme de flashs. Selon un mode particulier, l’éclairage est réalisé avec une lumière bleue (WO 2017/050917), plus particulièrement sous la forme d'un rayonnement présentant un spectre étroit de longueur d’onde comprise entre 400 et 550 nm, avantageusement un spectre étroit de longueur d’onde comprise entre 420 nm et 500 nm, avantageusement entre 430 et 480 nm, très préférentiellement centré sur 455 nm. Lighting may be continuous or discontinuous, in particular discontinuous in the form of flashes. According to a particular embodiment, the illumination is carried out with a blue light (WO 2017/050917), more particularly in the form of a radiation having a narrow spectrum of wavelength between 400 and 550 nm, advantageously a narrow spectrum wavelength between 420 nm and 500 nm, preferably between 430 and 480 nm, very preferably centered on 455 nm.
L’invention est caractérisée par le fait que l’on ajoute du glycérol au milieu de culture en quantité suffisante pour augmenter la production de phycocyanines par rapport à la culture sans glycérol.  The invention is characterized in that glycerol is added to the culture medium in an amount sufficient to increase the production of phycocyanins relative to the culture without glycerol.
Cet ajout peut être fait dans le milieu de culture en début de culture, ou encore en milieux de culture, une fois que la biomasse ait atteint une densité déterminée, par exemple supérieure à 20 g/L de matière sèche dans le milieu de culture.  This addition can be made in the culture medium at the beginning of culture, or in culture media, once the biomass has reached a specific density, for example greater than 20 g / l of dry matter in the culture medium.
L’étape (i) de culture peut donc se scinder en deux sous-étapes, (ia) de croissance pour produire de la biomasse avec une source de carbone comprenant essentiellement du glucose sous une forme simple ou complexe telle que définie précédemment et (ib) d’accumulation avec l’ajout de glycérol pour favoriser la production de phycocyanine.  The step (i) of culture can therefore be divided into two substeps, (ia) of growth to produce biomass with a carbon source comprising essentially glucose in a simple or complex form as defined above and (ib) ) of accumulation with the addition of glycerol to promote the production of phycocyanin.
Les sources de glycérol industrielles pouvant être intégrées dans les procédés de production n’ont pas de restriction sur la pureté ou le raffinage (généralement compris entre 80% et 100%). De préférence elles sont de grade alimentaire. Les fournisseurs en source de glycérol sont connus, en particulier des acteurs de l’industrie du biodiesel tel que le groupe Avril avec Oléon (Glycérine 4808/ 4808K, Glycérine 4827 / 4827K ...), ou la société Cargill qui propose également un large éventail de produit à base de glycérine raffinée entre 86.5% et 99.7% possédant des certifications casher, halal, RSPO, E442 ou sans OGM, notamment.  Industrial glycerol sources that can be integrated into production processes have no restriction on purity or refining (generally between 80% and 100%). Preferably they are food grade. Glycerol source suppliers are well known, particularly players in the biodiesel industry such as the Avril group with Oléon (Glycerine 4808 / 4808K, Glycerin 4827 / 4827K ...), or Cargill, which also offers a wide range of products. a product range based on glycerine refined between 86.5% and 99.7% possessing Kosher, Halal, RSPO, E442 or GMO-free certifications, among others.
La source de carbone comprenant du glucose est la principale source de carbone dans le milieu de culture et la quantité de glycérol suffisante pour augmenter la production de phycocyanine par rapport à la culture sans glycérol sera aisément déterminée par l’homme du métier par une simple expérimentation en fermenteurs permettant de comparer la quantité de phycocyanine produite avec ou sans glycérol comme montré dans les exemples.  The carbon source comprising glucose is the main source of carbon in the culture medium and the amount of glycerol sufficient to increase the production of phycocyanin relative to the culture without glycerol will be readily determined by those skilled in the art by a simple experiment. fermenters for comparing the amount of phycocyanin produced with or without glycerol as shown in the examples.
L’augmentation de production de phycocyanine recherchée sera préférentiellement d’au moins 0,15 mg/g/h, plus préférentiellement d’au moins 0,30 mg/g/h, encore plus préférentiellement d’au moins 1 mg/g/h ; ou bien d’au moins 3,6 mg/g, plus préférentiellement d’au moins 7,2 mg/g, encore plus préférentiellement d’au moins 24 mg/gX.  The desired increase in phycocyanin production will preferably be at least 0.15 mg / g / h, more preferably at least 0.30 mg / g / h, more preferably at least 1 mg / g / h; or at least 3.6 mg / g, more preferably at least 7.2 mg / g, more preferably at least 24 mg / gX.
En général, la quantité de glycérol ajoutée au milieu de culture est suffisante pour avoir un rapport pondéral glycérol /source de carbone comprenant le glucose (identifié par la suite par Gly/Glu) d’au moins 1/15. Préférentiellement le rapport Gly/Glu est d’au moins 1/14, plus préférentiellement d’au moins 1/10, encore plus préférentiellement d’au moins L’intérêt étant d’assurer une rentabilité optimum entre le coût des matières premières et la production de phycocyanine, il sera avantageux de ne pas dépasser un rapport Gly/Glu de 1/1. In general, the amount of glycerol added to the culture medium is sufficient to have a glycerol / carbon source weight ratio comprising glucose (hereinafter referred to as Gly / Glu) of at least 1/15. Preferably, the Gly / Glu ratio is at least 1/14, more preferably at least 1/10, even more preferably at least The interest is to ensure optimum profitability between the cost of raw materials and the production of phycocyanin, it will be advantageous not to exceed a Gly / Glu ratio of 1/1.
Selon un mode préféré de réalisation de l’invention, le glycérol est ajouté dans le milieu de culture pour un rapport pondéral Gly/Glu de 1/10 à 1/1 , préférentiellement de 1/7 à 1/2, plus préférentiellement d’environ 1/5 à environ 1/3.  According to a preferred embodiment of the invention, the glycerol is added to the culture medium for a Gly / Glu weight ratio of 1/10 to 1/1, preferably 1/7 to 1/2, more preferably about 1/5 to about 1/3.
Le procédé selon l'invention peut en outre comprendre une étape de récupération de la biomasse. Ladite récupération de la biomasse peut être réalisée par toute technique permettant la récupération de la biomasse, notamment les méthodes de filtration, gravimétrique ou sous pression réduite, de décantation, ou bien encore des méthodes de précipitation suivie d’une filtration gravimétrique.  The method according to the invention may further comprise a step of recovering the biomass. Said recovery of the biomass can be carried out by any technique allowing the recovery of biomass, including filtration methods, gravimetric or under reduced pressure, decantation, or else precipitation methods followed by gravimetric filtration.
L'invention concerne également la biomasse susceptible d'être obtenue par quelconque des variantes du procédé selon l'invention.  The invention also relates to the biomass obtainable by any of the variants of the process according to the invention.
On entend avantageusement par "biomasse" selon l’invention un ensemble de cellules de microorganismes produit par la culture de celles-ci, cellules qui peuvent avoir conservé ou non leur intégrité physique. On comprend donc que ladite biomasse peut comprendre une quantité de cellules de microorganismes dégradées allant de 0% à 100%. Par "dégradée" on entend que l’intégrité physique desdites cellules de microorganismes a pu être altérée comme par exemple des microorganismes lysés, résultant par exemple d’un procédé d’homogénéisation ou lyse enzymatique. Une fois produite, cette biomasse pourra être brute, juste séparée de son milieu de culture, séchée ou non, dégradée ou non.  The term "biomass" according to the invention is advantageously understood to mean a set of microorganism cells produced by the culture thereof, which cells may or may not have retained their physical integrity. It is thus understood that said biomass may comprise an amount of degraded microorganism cells ranging from 0% to 100%. By "degraded" it is meant that the physical integrity of said microorganism cells may have been altered such as, for example, lysed microorganisms, resulting for example from a homogenization or enzymatic lysis process. Once produced, this biomass can be raw, just separated from its culture medium, dried or not, degraded or not.
La biomasse, selon qu’elle soit séchée ou non, totalement ou en partie, peut comprendre un taux d’humidité de 1 % à 90%.  Biomass, depending on whether it is dried or not, in whole or in part, may comprise a moisture content of 1% to 90%.
Selon un premier mode de réalisation, la biomasse a un taux d’humidité de 70% à 90 %, préférentiellement 80% à 85 %. C’est en particulier le cas lorsqu’elle est essentiellement constituée de microorganismes industriels, optimisés et cultivés, après filtration du moût de fermentation pour séparer les microorganismes cultivés du milieu de culture, avant séchage.  According to a first embodiment, the biomass has a moisture content of 70% to 90%, preferably 80% to 85%. This is particularly the case when it consists essentially of industrial microorganisms, optimized and cultured, after filtration of the fermentation must to separate the cultured microorganisms from the culture medium, before drying.
Selon un autre mode de réalisation de l’invention, la biomasse est séchée, totalement ou en partie et présente un taux d’humidité de 1 % à 10%, préférentiellement de 2% à 7%. Selon l'invention ladite biomasse peut présenter une densité en ARUs comprise entre 20 et 200 g/l de matière sèche, préférentiellement entre 90 et 150 g/l de matière sèche.  According to another embodiment of the invention, the biomass is dried in whole or in part and has a moisture content of 1% to 10%, preferably 2% to 7%. According to the invention said biomass may have a density in ARUs of between 20 and 200 g / l of dry matter, preferably between 90 and 150 g / l of dry matter.
Selon l'invention ladite biomasse peut présenter une quantité de protéines comprise entre 25% et 60%, voire jusqu’à 70 %, de préférence entre 30% et 55%, plus préférentiellement entre 40% et 50% du poids de matière sèche. Le dosage de l'azote et le calcul de la teneur en protéines brutes sont réalisés selon la méthode de digestion en bloc et distillation à la vapeur (NF EN ISO 5983-2). According to the invention, said biomass may have a protein content of between 25% and 60%, even up to 70%, preferably between 30% and 55%, more preferably between 40% and 50% by weight of dry matter. The determination of nitrogen and the crude protein content is calculated using the bulk digestion method and steam distillation (NF EN ISO 5983-2).
Selon l'invention ladite biomasse peut présenter une teneur intracellulaire en phycobiliprotéines (phycocyanine et allophycocyanine) comprises entre 1 et 250 mg/g de matière sèche, préférentiellement entre 20 et 150 mg/g de matière sèche.  According to the invention said biomass may have an intracellular content of phycobiliproteins (phycocyanin and allophycocyanin) of between 1 and 250 mg / g of dry matter, preferably between 20 and 150 mg / g of dry matter.
Selon l'invention encore ladite biomasse peut présenter une teneur intracellulaire en phycocyanine comprise entre 0,5 et 100 mg/g de matière sèche, préférentiellement entre 10 et 40 mg/g de matière sèche.  According to the invention, said biomass may have an intracellular phycocyanin content of between 0.5 and 100 mg / g of dry matter, preferably between 10 and 40 mg / g of dry matter.
La biomasse pourra être conditionnée pour son stockage ou pour son utilisation en tant que telle, par exemple comme complément alimentaire ou aliment pour l’alimentation humaine ou animale.  Biomass may be conditioned for storage or for its use as such, for example as a food supplement or food for human or animal consumption.
Le tourteau susceptible d'être obtenu après extraction de la phycocyanine à partir de la biomasse d'ARUs susceptible d'être obtenue par le procédé selon l'invention peut être utilisé comme complément alimentaire riche en protéines et caroténoïdes, dans l’alimentation humaine ou animale.  The cake that can be obtained after extraction of the phycocyanin from the biomass of ARUs that can be obtained by the method according to the invention can be used as a dietary supplement rich in proteins and carotenoids, in human food or animal.
Selon l'invention ladite biomasse peut présenter une teneur intracellulaire en caroténoïdes comprise entre 0,1 et 10 mg/g de matière sèche, avantageusement entre 0,250 et 1 mg/g de matière sèche.  According to the invention said biomass may have an intracellular content of carotenoids of between 0.1 and 10 mg / g of dry matter, advantageously between 0.250 and 1 mg / g of dry matter.
Les ARUs présentent un potentiel d'utilisation important dans beaucoup de domaines dont on citera par exemple, l'alimentation humaine ou animale, la cosmétique, la médecine.  ARUs have a significant potential for use in many fields, such as, for example, human or animal nutrition, cosmetics and medicine.
Selon l'invention, ladite biomasse d'ARUs susceptible d'être obtenue selon l'invention peut être utilisée après récolte soit directement, éventuellement séchée, soit après transformation. En particulier ladite biomasse peut être utilisée sous la forme de farines entrant dans des compositions alimentaires ou sous forme de compléments alimentaires.  According to the invention, said biomass of ARUs obtainable according to the invention can be used after harvest either directly, optionally dried or after transformation. In particular, said biomass may be used in the form of flours entering food compositions or in the form of food supplements.
La biomasse d'ARUs susceptible d'être obtenue selon l'invention peut être transformée en farine selon tout procédé connu de l'homme du métier. On peut ainsi envisager par exemple que les ARUs puissent être séparées du milieu de culture, lysées et réduites en particules fines (diamètre moyen de 10 microns), puis séchées.  The biomass of ARUs that can be obtained according to the invention can be transformed into flour according to any method known to those skilled in the art. It can thus be envisaged, for example, that the ARUs can be separated from the culture medium, lysed and reduced to fine particles (average diameter of 10 microns), and then dried.
L'invention concerne également toute utilisation de la biomasse d'ARUs susceptible d'être obtenue selon l'invention dans tout domaine connu d'utilisation des ARUs, particulièrement, l'alimentation humaine ou animale, la cosmétique, la médecine. Dans les domaines de l'alimentation humaine ou animale et de la cosmétique, il s'agit bien évidement d'utilisations non thérapeutiques qui s'adressent à des animaux ou des êtres humains sains.  The invention also relates to any use of the biomass of ARUs obtainable according to the invention in any known field of use of ARUs, particularly, food or feed, cosmetics, medicine. In the fields of human or animal nutrition and cosmetics, these are of course non-therapeutic uses for animals or healthy humans.
La biomasse obtenue après culture de ARUs selon le procédé de l'invention peut permettre d'obtenir en particulier une farine riche en agents antioxydants, en particulier en caroténoïdes (particulièrement zéaxanthine et b-carotènes) en teneurs comprises entre 0,1 et 10 mg/g de matière sèche, avantageusement entre 0,25 et 1 mg/g de matière sèche dont en particulier de la zéaxanthine en une teneur comprise entre 0,05 et 5 mg/g de matière sèche, avantageusement entre 0,1 et 1 mg/g de matière sèche, et/ou du b- carotène en une teneur 0,05 et 5 mg/g de matière sèche, avantageusement entre 0,1 et 1 mg/g de matière sèche, répondant à un besoin en particulièrement dans l’industrie alimentaire, parce que plus appétentes, ayant meilleur goût, apportant des antioxydants en quantité importante et pouvant être utilisable dans l’alimentation animale ou humaine. The biomass obtained after culture of ARUs according to the method of the invention can make it possible to obtain in particular a flour rich in antioxidants, in particular carotenoids (particularly zeaxanthin and b-carotenes) in contents of between 0.1 and 10 mg / g of dry matter, advantageously between 0.25 and 1 mg / g of dry matter, in particular zeaxanthin in a content of between 0 and 10 mg / g. , 05 and 5 mg / g of dry matter, advantageously between 0.1 and 1 mg / g of dry matter, and / or of b-carotene at a content of 0.05 and 5 mg / g of dry matter, advantageously between 0 and , 1 and 1 mg / g of dry matter, meeting a need particularly in the food industry, because more palatable, having better taste, providing antioxidants in large quantities and can be used in animal or human food.
L'invention concerne donc une farine susceptible d'être obtenue après transformation de la biomasse en ARUs susceptible d'être obtenue par le procédé selon l'invention.  The invention therefore relates to a flour that can be obtained after transformation of the biomass into ARUs that can be obtained by the process according to the invention.
Quelle que soit la forme d'utilisation du produit susceptible d’être obtenu par le procédé selon l’invention (biomasse native ou transformée), ledit produit peut être utilisé pur ou mélangé à d’autres ingrédients classiquement utilisés, particulièrement dans des utilisations non thérapeutiques en alimentation ou en cosmétique.  Whatever the form of use of the product that can be obtained by the process according to the invention (native or transformed biomass), said product can be used pure or mixed with other conventionally used ingredients, particularly in non-organic uses. therapeutics in food or cosmetics.
L'invention concerne également tout produit pouvant comprendre au moins de la biomasse d'algues susceptible d'être obtenue selon l'invention. L'invention concerne aussi tout produit pouvant comprendre au moins de la farine issue de la transformation de la biomasse d'algues susceptible d'être obtenue selon l'invention.  The invention also relates to any product that may comprise at least the algal biomass obtainable according to the invention. The invention also relates to any product which may comprise at least flour resulting from the transformation of the algal biomass obtainable according to the invention.
Selon l'invention les phycocyanines produites par ladite biomasse peuvent être extraites pour être utilisées par exemple dans l'alimentation ou encore comme colorant. L'extraction des phycobiliprotéines, et particulièrement de la phycocyanine, à partir de ladite biomasse peut se faire selon toute technique d'extraction connue de l'homme du métier comme par exemple celle décrite par Moon & al. (2014) ou par Jaouen & al. (1999) ou dans les demandes FR 2 789 399, WO 2017/093345 et FR 1752674 déposée le 30 mars 2017.  According to the invention, the phycocyanins produced by said biomass can be extracted for use, for example, in the diet or as a dye. The extraction of phycobiliproteins, and particularly phycocyanin, from said biomass may be carried out according to any extraction technique known to those skilled in the art such as that described by Moon et al. (2014) or by Jaouen & al. (1999) or in the applications FR 2 789 399, WO 2017/093345 and FR 1752674 filed March 30, 2017.
L'invention concerne également l'utilisation de la phycocyanine susceptible d'être obtenue selon le procédé de l'invention, dans l'alimentation, animale ou humaine, comme complément alimentaire, ou encore comme colorant, particulièrement comme colorant alimentaire.  The invention also relates to the use of phycocyanin obtainable according to the method of the invention, in food, animal or human, as a dietary supplement, or as a dye, particularly as food coloring.
Elle concerne aussi un aliment comprenant la phycocyanine obtenue par le procédé selon l’invention, en particulier une boisson, plus particulièrement une boisson gazeuse.  It also relates to a feed comprising the phycocyanin obtained by the process according to the invention, in particular a beverage, more particularly a gaseous drink.
EXEMPLES EXAMPLES
Exemple 1 : Comparatif de croissance de la souche de G. sulphuraria sur glycérol, glucose et un mélange glucose-glycérol.  Example 1: Comparative growth of the G. sulphuraria strain on glycerol, glucose and a glucose-glycerol mixture.
La croissance de la souche est réalisée par mesure de l’absorbance à 800nm au cours du temps. Le milieu de culture est celui connu pour cette souche (milieu Gross), la seule différence entre les trois milieux testés est le ou les substrat(s) carboné(s) utilisé(s).The growth of the strain is carried out by measuring the absorbance at 800 nm over time. The culture medium is the one known for this strain (medium Gross), the the only difference between the three media tested is the carbon substrate (s) used.
M&M : Souche : Galdieria sulphuraria (aussi appelée Cyanidium caldarium) UTEX#2919 M & M: Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX # 2919
Milieu de culture : 30g/L de glucose ou 30g/L de glycérol (glycérine 4808 Univar) ou 25g/L de glucose + 5g/L de glycérol ; 8g/L de (NH4)2S04 ; 0,25g/L de KH2P04 ; 716mg/L de MgS04 ; 44mg/L CaCI2 ; 3ml/L de solution stock de Fe-EDTA (FeS04 à 6,9g/L et Na2-EDTA à 9,3g/L) et 4ml de solution stock de trace métal (3,09g/L de Na2- EDTA ; 0,08g/L CuS04,5H20 ; 2,86g/L H3B03 ; 0,04g/L NaV03,4H20 ; 1 ,82g/L MnCI2 ; 0,04g/L CoCI2,6H20 ; 0,22g/L ZnS04,7H20 ; 0,017g/L Na2Se03 ; 0,03g/L (NH4)6Mo7024, 4H20).  Culture medium: 30g / L of glucose or 30g / L of glycerol (glycerin 4808 Univar) or 25g / L of glucose + 5g / L of glycerol; 8 g / L of (NH4) 2SO4; 0.25 g / L of KH 2 PO 4; 716 mg / L MgSO4; 44mg / L CaCl2; 3 ml / L of Fe-EDTA stock solution (FeS04 at 6.9 g / L and Na2-EDTA at 9.3 g / L) and 4 ml of metal trace stock solution (3.09 g / L of Na2-EDTA; 08g / L CuS04.5H2O, 2.86 g / L H3B03, 0.04g / L NaV03.4H2O, 1.82g / L MnCl2, 0.04g / L CoCl2.6H2O, 0.22g / L ZnSO4.7H2O, 0.017g / L Na2SeO3, 0.03g / L (NH4) 6Mo7O4, 4H2O).
Conditions de culture : Les cultures sont réalisées sur une table d’agitation (140t/min) en enceinte thermostatée à 37°C, en milieu comme décrit ci-dessus en présence d’une source de lumière.  Culture conditions: The cultures are carried out on a stirring table (140 rpm) in a thermostatically controlled enclosure at 37 ° C. in the middle as described above in the presence of a light source.
Suivi des cultures : Le suivi de croissance montre que l’on atteint des DO similaires quelque soit la ou les source(s) de carbone utilisée(s), les masses sèches et les dosages en phycocyanine associés au bout de 312h de culture sont décrits dans le tableau.  Crop monitoring: The growth monitoring shows that we achieve similar OD regardless of the source (s) of carbon (s) used, dry masses and associated phycocyanin assays after 312h of culture are described In the picture.
L’apport de glycérol dans le milieu active la production de phycocyanine malgré la présence du glucose connu comme inhibiteur de la phycocyanine. The addition of glycerol in the medium activates the production of phycocyanin despite the presence of glucose known as phycocyanin inhibitor.
Exemple 2 : Comparatif de croissance de la souche de G. sulphuraria sur perméat de lait et un mélange perméat de lait-glycérol.  EXAMPLE 2 Comparison of growth of the G. sulphuraria strain on milk permeate and a permeate mixture of milk-glycerol.
La croissance de la souche est réalisée par mesure de l’absorbance à 800nm au cours du temps. Le milieu de culture est celui connu pour cette souche (milieu Gross), la seule différence entre les deux milieux testés est le ou les substrat(s) carboné(s) utilisé(s).  The growth of the strain is carried out by measuring the absorbance at 800 nm over time. The culture medium is that known for this strain (medium Gross), the only difference between the two media tested is the carbon substrate (s) used (s).
M&M : Souche : Galdieria sulphuraria (aussi appelée Cyanidium caldarium) UTEX#2919  M & M: Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX # 2919
Milieu de culture : 30g/L de perméat de lait ou 25g/L de perméat de lait + 5g/L de glycérol (glycérine 4808 Univar) ; 8g/L de (NH4)2S04 ; 0,25g/L de KH2P04 ; 716mg/L de MgS04 ; 44mg/L CaCI2 ; 3ml/L de solution stock de Fe-EDTA (FeS04 à 6,9g/L et Na2- EDTA à 9,3g/L) et 4ml de solution stock de trace métal (3,09g/L de Na2-EDTA ; 0,08g/L CuS04,5H20 ; 2,86g/L H3B03 ; 0,04g/L NaV03,4H20 ; 1 ,82g/L MnCI2 ; 0,04g/L CoCI2,6H20 ; 0,22g/L ZnS04,7H20 ; O,017g/L Na2Se03 ; 0,03g/L (NH4)6Mo7024, 4H20). Culture medium: 30g / L of milk permeate or 25g / L of milk permeate + 5g / L of glycerol (glycerin 4808 Univar); 8 g / L of (NH4) 2SO4; 0.25 g / L of KH 2 PO 4; 716 mg / L MgSO4; 44mg / L CaCl2; 3 ml / L of Fe-EDTA stock solution (FeS04 at 6.9g / L and Na2-EDTA at 9.3 g / L) and 4 ml of metal trace stock solution (3.09 g / L Na2-EDTA; 08g / L CuS04.5H2O, 2.86g / L H3B03, 0.04g / L NaV03.4H2O, 1.82g / L MnCl2, 0.04g / L CoCl2.6H2O; 0.22g / L ZnSO4.7H2O; 0, 017g / L Na2SeO3; 0.03 g / L (NH4) 6Mo7024, 4H2O).
Conditions de culture : Les cultures sont réalisées sur une table d’agitation (140t/min) en enceinte thermostatée à 37°C, en milieu comme décrit ci-dessus en présence ou non d’une source de lumière.  Culture conditions: The cultures are carried out on a stirring table (140 rpm) in a thermostatically controlled enclosure at 37 ° C., in the medium as described above, in the presence or absence of a light source.
Suivi des cultures : Les masses sèches et les dosages en phycocyanine associés au bout de 168h de culture sont décrits dans le tableau 2(A) en présence de lumière et dans le tableau 2(B) à l’obscurité.  Crop Monitoring: The dry masses and associated phycocyanin assays after 168 h of culture are described in Table 2 (A) in the presence of light and in Table 2 (B) in the dark.
L’apport de glycérol dans le milieu active la production de phycocyanine malgré la présence du perméat de lait inhibiteur de la phycocyanine et cette augmentation de la production se remarque même à l’obscurité. The presence of glycerol in the medium activates the production of phycocyanin despite the presence of phycocyanin-inhibiting milk permeate and this increase in production is noticeable even in the dark.
Exemple 3 : Suivi d’un procédé de culture en bioréacteur de la souche de G.sulphuraria sur un mélange glucose-glycérol.  Example 3: Follow-up of a bioreactor culture method of the G.sulphuraria strain on a glucose-glycerol mixture.
Le suivi de croissance de la souche est réalisé par mesure de la masse sèche au cours du temps et la consommation en substrat est identifiée par dosage HPLC. Un dosage de la phycocyanine intracellulaire est également réalisé au cours du temps. Le milieu de culture est celui connu pour cette souche (milieu Gross) avec pour substrat 1/6 de glycérol et 5/6 de glucose pour le pied de cuve et une alimentation composée de ¼ de glycérol et ¾ de glucose pour le fed-batch.  The growth of the strain is monitored by measuring the dry mass over time and the substrate consumption is identified by HPLC assay. An intracellular phycocyanin assay is also performed over time. The culture medium is that known for this strain (medium Gross) with 1/6 substrate of glycerol and 5/6 of glucose for the stock and a diet consisting of ¼ glycerol and ¾ glucose for the fed-batch .
M&M : Souche : Galdieria sulphuraria (aussi appelée Cyanidium caldarium) UTEX#2919  M & M: Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX # 2919
Milieu de culture :  Culture centre :
Pied de cuve : 25g/L glucose + 5g/L de glycérol (glycérine 4808 Univar) ; 8g/L de Foot of tank: 25g / L glucose + 5g / L of glycerol (glycerin 4808 Univar); 8g / L of
(NH4)2S04 ; 0,25g/L de KH2P04 ; 716mg/L de MgS04 ; 44mg/L CaCI2 ; 3ml/L de solution stock de Fe-EDTA (FeS04 à 6,9g/L et Na2-EDTA à 9,3g/L) et 4ml de solution stock de trace métal (3,09g/L de Na2-EDTA ; 0,08g/L CuS04,5H20 ; 2,86g/L H3B03 ; 0,04g/L NaV03,4H20 ; 1 ,82g/L MnCI2 ; 0,04g/L CoCI2,6H20 ; 0,22g/L ZnS04,7H20 ; 0,017g/L Na2Se03 ; 0,03g/L (NH4)6Mo7024, 4H20). (NH4) 2SO4; 0.25 g / L of KH 2 PO 4; 716 mg / L MgSO4; 44mg / L CaCl2; 3ml / L of stock solution of Fe-EDTA (FeS04 at 6.9g / L and Na2-EDTA at 9.3g / L) and 4ml of metal trace stock solution (3.09g / L Na2-EDTA, 0.08g / L CuS04 , 5H20, 2.86 g / L H3B03, 0.04 g / L NaV03.4H2O, 1.82 g / L MnCl2, 0.04 g / L CoCl2.6H2O, 0.22 g / L ZnSO4.7H2O, 0.017 g / L Na2SeO3; 0.03 g / L (NH4) 6Mo7024, 4H2O).
Milieu Fed-Batch : 375g/L glucose + 125g/L de glycérol (glycérine 4808 Univar) ; 20g/L de (NH4)2S04 ; 4,17g/L de KH2P04 ; 5,96 g/L de MgS04 ; 0,37 g/L CaCI2 ; 25ml/L de solution stock de Fe-EDTA (FeS04 à 6,9g/L et Na2-EDTA à 9,3g/L) et 33ml de solution stock de trace métal (3,09g/L de Na2-EDTA ; 0,08g/L CuS04,5H20 ; 2,86g/L H3B03 ; 0,04g/L NaV03,4H20 ; 1 ,82g/L MnCI2 ; 0,04g/L CoCI2,6H20 ; 0,22g/L ZnS04,7H20 ; 0,017g/L Na2Se03 ; 0,03g/L (NH4)6Mo7024, 4H20).  Fed-Batch medium: 375g / L glucose + 125g / L glycerol (glycerin 4808 Univar); 20 g / L of (NH4) 2SO4; 4.17 g / L of KH 2 PO 4; 5.96 g / L MgSO4; 0.37 g / L CaCl 2; 25 ml / L of Fe-EDTA stock solution (FeS04 at 6.9g / L and Na2-EDTA at 9.3 g / L) and 33 ml of metal trace stock solution (3.09 g / L of Na2-EDTA; 08g / L CuS04.5H2O, 2.86 g / L H3B03, 0.04g / L NaV03.4H2O, 1.82g / L MnCl2, 0.04g / L CoCl2.6H2O, 0.22g / L ZnSO4.7H2O, 0.017g / L Na2SeO3, 0.03g / L (NH4) 6Mo7O4, 4H2O).
Conditions de culture : Les cultures sont réalisées en bioréacteur de 2L à une température régulée à 37°C, pH 3, en présence d’une source de lumière.  Culture conditions: The cultures are carried out in a 2L bioreactor at a temperature controlled at 37 ° C., pH 3, in the presence of a light source.
Le suivi de croissance montre que l’on atteint une masse sèche de 70g/l au bout de 427h de culture et une teneur en phycocyanine intracellulaire de 45mg/gX (Fig. 1 ). La totalité du substrat carboné apportée est consommée.  Growth monitoring shows that a dry mass of 70 g / l is reached after 427 h of culture and an intracellular phycocyanin content of 45 mg / g × (Fig. 1). The entire carbon substrate brought is consumed.
Exemple 4 : Suivi d’un procédé de culture en bioréacteur de la souche de G.sulphuraria sur un mélange perméat de lait-glycérol.  Example 4: Follow-up of a bioreactor culture method of the G.sulphuraria strain on a permeate mixture of milk-glycerol.
Le suivi de croissance de la souche est réalisé par mesure de la masse sèche au cours du temps et la consommation en substrat est identifié par dosage HPLC. Un dosage de la phycocyanine intracellulaire est également réalisé au cours du temps. Le milieu de culture est celui connu pour cette souche (milieu Gross) avec pour substrat 1/6 de glycérol et 5/6 de lactose (apporté par le perméat de lait) pour le pied de cuve et une alimentation composée de ¼ de glycérol et ¾ de lactose pour le fed-batch.  The growth of the strain is monitored by measuring the dry mass over time and the substrate consumption is identified by HPLC assay. An intracellular phycocyanin assay is also performed over time. The culture medium is the one known for this strain (medium Gross) with for 1/6 substrate of glycerol and 5/6 of lactose (brought by the permeate of milk) for the base of vat and a feed composed of ¼ glycerol and ¾ of lactose for fed-batch.
M&M : Souche : Galdieria sulphuraria (aussi appelée Cyanidium caldarium) UTEX#2919  M & M: Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX # 2919
Milieu de culture :  Culture centre :
Pied de cuve : 25g/L perméat de lait + 5g/L de glycérol (glycérine 4808 Univar) ; 8g/L de (NH4)2S04 ; 716mg/L de MgS04 ; 3ml/L de solution stock de Fe-EDTA (FeS04 à 6,9g/L et Na2-EDTA à 9,3g/L) et 4ml de solution stock de trace métal (3,09g/L de Na2- EDTA ; 0,08g/L CuS04,5H20 ; 2,86g/L H3B03 ; 0,04g/L NaV03,4H20 ; 1 ,82g/L MnCI2 ; 0,04g/L CoCI2,6H20 ; 0,22g/L ZnS04,7H20 ; 0,017g/L Na2Se03 ; 0,03g/L Foot of tank: 25g / L permeate of milk + 5g / L of glycerol (Glycerin 4808 Univar); 8 g / L of (NH4) 2SO4; 716 mg / L MgSO4; 3 ml / L of Fe-EDTA stock solution (FeS04 at 6.9 g / L and Na2-EDTA at 9.3 g / L) and 4 ml of metal trace stock solution (3.09 g / L of Na2-EDTA; 08g / L CuS04.5H2O, 2.86 g / L H3B03, 0.04g / L NaV03.4H2O, 1.82g / L MnCl2, 0.04g / L CoCl2.6H2O, 0.22g / L ZnSO4.7H2O, 0.017g / L Na2SeO3 0.03g / L
(NH4)6Mo7024, 4H20). (NH4) 6Mo7024, 4H2O).
Milieu Fed-Batch : 85g/L de perméat de lait ; 28,3g/L de glycérol  Fed-Batch medium: 85g / L of milk permeate; 28.3g / L of glycerol
Conditions de culture : Les cultures sont réalisées en bioréacteur de 2L à une température régulée à 37°C, pH 3, en présence d’une source de lumière.  Culture conditions: The cultures are carried out in a 2L bioreactor at a temperature controlled at 37 ° C., pH 3, in the presence of a light source.
Le suivi de croissance montre que l’on atteint une masse sèche de 25g/l au bout de 450h de culture et une teneur en phycocyanine intracellulaire de 14mg/g.  Growth monitoring shows that a dry mass of 25 g / l is reached after 450 h of culture and an intracellular phycocyanin content of 14 mg / g.
Exemple 5 : Variation de la production de PC en fonction de la proportion Gly/GIc dans le milieu de culture. Example 5: Variation of PC Production by Gly / GIc Proportion in the culture medium.
Une gamme croisée de glucose et de glycérol a été réalisée pour permettre d'évaluer l’effet du glycérol sur la levée d’inhibition de la synthèse de phycocyanine liée à la présence de glucose dans le milieu de culture. La quantité totale de carbone organique dans chaque Erlenmeyer est de 30 g/l. Le glucose est symbolisé par la contraction « glc » et le glycérol par « gly ». La concentration de chaque élément dans le milieu est indiquée dans le tableau 3.  A cross-over range of glucose and glycerol was performed to evaluate the effect of glycerol on the inhibition of inhibition of phycocyanin synthesis related to the presence of glucose in the culture medium. The total amount of organic carbon in each Erlenmeyer flask is 30 g / l. Glucose is symbolized by the contraction "glc" and glycerol by "gly". The concentration of each element in the medium is shown in Table 3.
Production de PC selon les substrats (mg/gX/h)  PC production by substrates (mg / gX / h)
Exemple 6 : Variation de la production de PC en fonction de la proportion Gly/GIc dans le milieu de culture (Concentration en glycérol inférieure à 5g/l). EXAMPLE 6 Variation of the PC Production as a Function of the Gly / GIc Proportion in the Culture Medium (Glycerol concentration less than 5 g / l)
Cet exemple vient compléter l’exemple 5, les conditions de culture y sont similaires la seule différence est au niveau des concentrations en glycérol testées qui sont inférieure ou égale à 5g/l afin de quantifier la concentration en glycérol limite permettant de lever l'inhibition de la synthèse de phycocyanine par le glucose dans le milieu de culture.  This example complements the example 5, the culture conditions are similar the only difference is in the glycerol concentrations tested which are less than or equal to 5g / l in order to quantify the limit glycerol concentration to lift the inhibition of phycocyanin synthesis by glucose in the culture medium.
Productivité (mg/g/h)  Productivity (mg / g / h)
Exemple 7 : Variation de la production de PC en fonction de la proportion Glycérol apporté dans un milieu contenant en début de culture 30g/l de perméat de lait. Example 7 Variation of PC Production by Glycerol Proportion brought into a medium containing at the beginning of the culture 30g / l of milk permeate.
Différentes quantités de glycérol sont apportées à un milieu composé de perméat de lait afin d’observer l’effet de celui-ci sur la synthèse de phycocyanine et la levée de l’inhibition du lactose (glucose + saccharose) sur la production de ce pigment. Le milieu de base contient 30g/l de perméat de lait. Le glycérol ajouté est symbolisé par la contraction « gly ». 6 concentrations en glycérol sont testées (0, 1 , 2, 3, 4 et 5g/l).  Various amounts of glycerol are added to a medium composed of milk permeate in order to observe the effect of this on the phycocyanin synthesis and the removal of the lactose (glucose + sucrose) inhibition on the production of this pigment . The base medium contains 30g / l of milk permeate. The added glycerol is symbolized by the contraction "gly". 6 glycerol concentrations are tested (0, 1, 2, 3, 4 and 5g / l).
Productivité (mg/L/h)  Productivity (mg / L / h)
Similairement, a ce qui a été décrit Stadnichuk et al., (1998), le glucose a bien un effet limitant sur la quantité de phycocyanine produite par la souche de Galdieria sulphuraria, avec une concentration de moins de 15 mg de phycocyanine par g de matière sèche en fin de croissance (15 mg PC/g de MS). A l’opposé, la souche ayant poussée en présence de glycérol uniquement, accumule environ 40 mg PC/g de MS, pour le même temps de culture (240h). En prolongeant, la culture ayant poussé en glycérol peut avoir accumulé jusqu'à 70 mg de PC/g de MS, alors que celle ayant poussé en glucose aura une quantité de PC/g de MS qui restera plus ou moins constante dans le temps. Dans tous les autres cas (mélange glucose-glycérol) la biomasse produite ainsi que la quantité de PC présente dans la biomasse reste constante et même supérieure à celle du glycérol pour le même temps de culture. Ces valeurs sont comprises entre 50 et 60 mg de PC/g de MS, plus de trois fois supérieur à ce qui est trouvé dans la biomasse de cellules ayant poussé sur glucose uniquement. Similarly, according to Stadnichuk et al., (1998), glucose has a limiting effect on the amount of phycocyanin produced by the Galdieria sulphuraria strain, with a concentration of less than 15 mg of phycocyanin per g of dry matter at the end of growth (15 mg PC / g of MS). In contrast, the strain having grown in the presence of glycerol only, accumulates about 40 mg PC / g of MS, for the same culture time (240h). By prolonging, the culture grown in glycerol may have accumulated up to 70 mg of PC / g of MS, while the one having grown in glucose will have a quantity of PC / g of MS which will remain more or less constant over time. In all other cases (glucose-glycerol mixture) the biomass produced as well as the amount of PC present in the biomass remains constant and even greater than that of glycerol for the same culture time. These values are between 50 and 60 mg PC / g DM, more than three times higher than what is found in the cell biomass grown on glucose alone.
En conclusion, l’addition de petites quantités de glycérol dans le milieu permet de lever l’inhibition de la synthèse de PC liée à la présence de glucose dans le milieu, qu’il soit présent sous forme de sucre simple (glucose), ou complexe (lactose, saccharose, ou autre ...).  In conclusion, the addition of small amounts of glycerol in the medium makes it possible to eliminate the inhibition of PC synthesis linked to the presence of glucose in the medium, whether it is present in the form of simple sugar (glucose), or complex (lactose, sucrose, or other ...).
Ce procédé permet de faire croître les cellules en utilisant le glucose (ou une source de carbone contenant du glucose) comme source de carbone principal, et d’utiliser le glycérol uniquement comme inducteur de synthèse de PC et non pas comme substrat principal. Le glucose ou les sources de carbones contenant du glucose sont en général moins chers que le glycérol, et permettent en général d’obtenir des taux de croissance plus élevés que sur glycérol, donc une économie sur les coûts de fermentation. This method makes it possible to grow the cells using glucose (or a carbon source containing glucose) as the main carbon source, and to use glycerol only as a PC synthesis inducer and not as a main substrate. Glucose or carbon sources containing glucose are usually cheaper than glycerol, and generally allow to obtain growth rates higher than glycerol, thus saving on fermentation costs.
REFERENCES REFERENCES
- Carfagna & al., Algal Research 31 (2018) 406-412  - Carfagna & al., Algal Research 31 (2018) 406-412
- Gross & al., Plant cell Physiol. 36 (1995) 633-638 ;  - Gross et al., Plant cell Physiol. 36 (1995) 633-638;
- Jaouen & al., Biotechnology techniques 13 (1999) 877-881  - Jaouen et al., Biotechnology Techniques 13 (1999) 877-881
- Moon & al., Korean J. Chem. Eng. 31 (2014) 490-495 ; - Moon & al., Korean J. Chem. Eng. 31 (2014) 490-495;
- Oesterhelt & al., Plant J. 51 (2007) 500-51 1  - Oesterhelt et al., Plant J. 51 (2007) 500-51 1
- Sloth & al., Enzyme and Microbial Technology 38 (2006) 168-175  Sloth et al., Enzyme and Microbial Technology 38 (2006) 168-175
- Stadnichuk & al., Plant Science 136 (1998) 1 1-23  - Stadnichuk et al., Plant Science 136 (1998) 1 1-23
- FR 2 789 399  - FR 2 789 399
- FR 3 064 635 - FR 3 064 635
- WO 2017/050917,  - WO 2017/050917,
- WO 2017/050918,  - WO 2017/050918,
- WO 2017/093345  - WO 2017/093345
- WO 2018/178334  - WO 2018/178334

Claims

REVENDICATIONS
1. Procédé de culture d’algues rouges unicellulaires (ARU) pour la production d’une biomasse riche en phycocyanines, comprenant les étapes (i) de cultures en mode mixotrophe ou hétérotrophe desdites ARU sur un milieu de culture comprenant une source de carbone comprenant du glucose, et (ii) de récupération de la biomasse, caractérisé en ce que l’on ajoute au milieu de culture une quantité de glycérol suffisante pour augmenter la production de phycocyanine par rapport à la culture sans glycérol.  A method of cultivating unicellular red algae (ARU) for the production of a biomass rich in phycocyanins, comprising the steps of (i) mixing in the mixotrophic or heterotrophic mode of said ARUs on a culture medium comprising a carbon source comprising glucose, and (ii) recovery of biomass, characterized in that there is added to the culture medium a sufficient amount of glycerol to increase the production of phycocyanin relative to the culture without glycerol.
2. Procédé selon la revendicaiton 1 , caractérisé en ce que les ARU sont les genres Cyanidioschyzon, Cyanidium ou Galdieria, en particulier parmi les espèces Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201 , Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita ou encore Galdieria sulphuraria.  2. Method according to revendicaiton 1, characterized in that the ARU are genera Cyanidioschyzon, Cyanidium or Galdieria, in particular among the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens , Galdieria daedala, Galdieria maxima, Galdieria partita or Galdieria sulphuraria.
3. Procédé selon l’une des revendications 1 ou 2, caractérisé en ce que la source de carbone comprenant du glucose est choisie parmi le glucose et le glucose sous une forme complexe comme le lactose, le fructose ou des polyosides comprenant du glucose, et leurs mélanges.  3. Method according to one of claims 1 or 2, characterized in that the carbon source comprising glucose is selected from glucose and glucose in a complex form such as lactose, fructose or polysaccharides comprising glucose, and their mixtures.
4. Procédé selon la revendication 3, caractérisé en ce que la source de carbone est du perméat de lait comprenant du lactose.  4. Method according to claim 3, characterized in that the carbon source is milk permeate comprising lactose.
5. Procédé selon l’une des revendications 1 à 4, caractérisé en ce que le milieu de culture comprend entre 0,05 g/L et 200 g/L de source de carbone comprenant du glucose.  5. Method according to one of claims 1 to 4, characterized in that the culture medium comprises between 0.05 g / L and 200 g / L of carbon source comprising glucose.
6. Procédé selon l’une des revendications 1 à 5, caractérisé en ce que l’étape de culture est en mode mixotrophe avec éclairage continu ou discontinu.  6. Method according to one of claims 1 to 5, characterized in that the culture step is in mixotrophic mode with continuous or discontinuous illumination.
7. Procédé selon l’une des revendications 1 à 6, caractérisé en ce que l’ajout de glycérol dans le milieu de culture est réalisé en début de culture ou en milieux de culture une fois que la biomasse ait atteint une densité déterminée.  7. Method according to one of claims 1 to 6, characterized in that the addition of glycerol in the culture medium is carried out at the beginning of culture or in culture media once the biomass has reached a specific density.
8. Procédé selon l’une des revendications 1 à 7, caractérisé en ce que la quantité de glycérol ajoutée au milieu de culture est suffisante pour avoir un rapport pondéral glycérol /source de carbone comprenant le glucose (Gly/Glu) d’au moins 1/15.  8. Method according to one of claims 1 to 7, characterized in that the amount of glycerol added to the culture medium is sufficient to have a weight ratio glycerol / carbon source comprising glucose (Gly / Glu) at least 1/15.
9. Procédé selon la revendication 8, caractérisé en ce que le rapport pondéral glycérol /source de carbone comprenant le glucose (Gly/Glu) est d’au moins 1/14.  9. The method of claim 8, characterized in that the weight ratio glycerol / carbon source comprising glucose (Gly / Glu) is at least 1/14.
10. Procédé selon la revendication 8, caractérisé en ce que le rapport pondéral glycérol /source de carbone comprenant le glucose (Gly/Glu) est d’au moins 1/10.  10. The method of claim 8, characterized in that the weight ratio glycerol / carbon source comprising glucose (Gly / Glu) is at least 1/10.
1 1. Procédé selon la revendication 8, caractérisé en ce que le rapport pondéral glycérol /source de carbone comprenant le glucose (Gly/Glu) est d’au moins 1/5.  1. Process according to claim 8, characterized in that the glycerol / carbon source weight ratio comprising glucose (Gly / Glu) is at least 1/5.
12. Procédé selon la revendication 8, caractérisé en ce que le rapport pondéral Gly/Glu va de 1/5 à 1/3. 12. The method of claim 8, characterized in that the weight ratio Gly / Glu is 1/5 to 1/3.
13. Procédé de préparation de phycocyanines caractérisé en ce qu’il comprend la production d’une biomasse selon l’une des revendications 1 à 12 et une étape (iii) d’extraction des phycocyanines à partir de la biomasse préalablement récupérée. 13. Process for the preparation of phycocyanines, characterized in that it comprises the production of a biomass according to one of claims 1 to 12 and a step (iii) of extracting phycocyanins from the previously recovered biomass.
14. Biomasse obtenue par le procédé selon l'une quelconque des revendications 1 à 13.  14. Biomass obtained by the process according to any one of claims 1 to 13.
15. Aliment caractérisée en ce qu’elle comprend une biomasse selon la revendication 14 ou une phycocyanine obtenue par le procédé selon la revendication 13.  15. A food characterized in that it comprises a biomass according to claim 14 or a phycocyanin obtained by the process according to claim 13.
EP19725747.0A 2018-05-31 2019-05-27 Method for cultivating unicellular red algae (ura) on a mixture of substrates Pending EP3802782A1 (en)

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