Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/009—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from unicellular algae
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
  • A23J3/347—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of proteins from microorganisms or unicellular 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
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/20—Synthetic spices, flavouring agents or condiments
  • A23L27/23—Synthetic spices, flavouring agents or condiments containing nucleotides
  • A23L27/235—Synthetic spices, flavouring agents or condiments containing nucleotides containing also amino acids
• • 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
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/88—Taste or flavour enhancing agents
• • 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
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/18—Peptides; Protein hydrolysates
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
• • 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
  • C12N1/02—Separating microorganisms from their culture media
• • 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
  • C12N1/06—Lysis of microorganisms
  • C12N1/066—Lysis of microorganisms by physical methods
• • 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
• • 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
  • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• • 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
  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

Definitions

• the present invention relates to a method for fractionating biomass components of microalgae rich in proteins.
• chlorella is a potential source of food because it is rich in protein and other essential nutrients.
• microalgae proteins Given their abundance and their amino acid profile, microalgae proteins are thus considered as an alternative source of soy protein or peas in Food.
• the protein fraction can also be promoted as a functional agent in the cosmetic or even pharmaceutical industries.
• these proteins must be extracted from microalgae without impacting their molecular structure.
• an efficient disintegration process preserving the integrity of the cellular components must maximize not only the yield but also the quality of the extracted products.
• an optimized wall disintegration method must for example avoid:
• the cells are agitated in suspension with small spherical particles.
• the breaking of the cells is caused by shear forces, grinding between the balls, and collisions with beads.
• This emulsion is generally atomized and the water is removed, leaving a dry powder containing however a heterogeneous mixture consisting of cell debris, interstitial soluble compounds and oil.
• the difficulty to solve in the use of these cell disruption technologies is the isolation of the only intracellular content (excluding membrane debris, sugars, fibers and fats) and the preservation, inter alia, of the quality protein load.
• the exposure of biological cells to a pulsed electric field of high intensity can indeed alter the structure of the cell membrane.
• the external field causes the loading of the membrane.
• a sufficient transmembrane voltage 0.5-1 V
• the molecular arrangement of the phospholipids changes, which leads to the membrane losing its role as a barrier, making it permeable.
• this membrane permeabilization may be reversible or irreversible.
• This rupture of the membrane then facilitates the release of the cellular contents and, when using a complementary solvent extraction technique, also facilitates the penetration of the solvent into the cell.
• the Applicant Company has found that this need could be satisfied by combining a thermal permeabilization process of the microalgae cells with centrifugation and precipitation steps by modulating the properties of the medium.
• the Applicant company thus goes against a technical prejudice that thermal methods of cell disruption, like the shear forces caused by mechanical disintegration, are rather implemented technologies for degrading or denaturing products derived from microalgae. (Richmond, 1986, Handbook of Microalgal Mass Culture. CRC Press, Inc. - Molina Grima et al., 2003, Recovery of Microalgal Biomass and Metabolites: Process Options and economiess, Biotechnol. Adv. 20: 491-515).
• the present invention therefore relates to a method of fractionating biomass components of microalgae rich in proteins:
• the process according to the invention is a process for fractionating the components of a biomass of microalgae rich in proteins of the Chlorella genus, characterized in that it comprises the following steps:
• thermal permeabilization of the biomass at a temperature between 50 and 150 ° C, preferably between about 80 and 150 ° C, for a period of between about 10 seconds and about 5 minutes, preferably for a period of time between about 5 seconds and about 1 minute, separation between the biomass thus permeabilized and the soluble fraction by a centrifugation technique, more particularly multi-stage centrifugation, - optionally, recovery and clarification of the soluble fraction thus obtained by microfiltration so as to rid it of residual insolubles, purification of the preceding soluble fraction by precipitation, in order to obtain a peptide isolate and a peptide concentrate.
• a centrifugation technique more particularly multi-stage centrifugation, - optionally, recovery and clarification of the soluble fraction thus obtained by microfiltration so as to rid it of residual insolubles, purification of the preceding soluble fraction by precipitation, in order to obtain a peptide isolate and a peptide concentrate.
• microalgae of the genus Chlorella are selected from the group consisting of Chlorella vulgaris, Chlorella sorokiniana and Chlorella protothecoides, and are more particularly Chlorella protothecoides.
• the strain is Chlorella protothecoides
• strain UTEX 250 The Culture Collection of Algae at the University of Texas at Austin - USA.
• the strain is CCAP21 strain 1 / 8D - The Culture Collection of Algae and Protozoa, Scotland, UK).
• Cultivation under heterotrophic conditions and in the absence of light typically leads to the production of a biomass of chbrelles having a protein content (evaluated by measurement of the nitrogen content N x 6.25) of 45 to 70% by weight. dry cell weight.
• a biomass of microalgae enriched in proteins for example having a protein content, expressed in N 6.25 greater than 60%.
• the applicant company recommends using an original process that it has developed, and which includes:
• the biomass is then collected by solid-liquid separation, by frontal or tangential filtration or by any other means known to those skilled in the art.
• the applicant company then recommends washing the biomass so as to eliminate the interstitial soluble compounds by a succession of concentration (by centrifugation) / dilution of the biomass.
• interstitial soluble compounds means all the soluble organic contaminants of the fermentation medium, for example the water-soluble compounds such as salts, residual glucose, oligosaccharides of degree of polymerization (or DP) 2 or 3 or the peptides.
• This biomass thus purified of its interstitial soluble compounds is then adjusted preferably to a solids content of between 15 and 30% by weight, preferably at a solids content of between 20 and 30%.
• the heat treatment is then carried out at a temperature of between 50 and 150 ° C., preferably between approximately 80 and 150 ° C., for a duration of between approximately 10 seconds and approximately 5 minutes, preferably for a duration of between approximately 5 seconds. and about 5 minutes, preferably for about 10 seconds to about 1 minute.
• the heat treatment is conducted at a temperature of about 140 ° C for about 10 seconds.
• the heat treatment is conducted at a temperature of about 85 ° C for about 1 minute.
• This treatment makes it possible to allow the intracellular components to diffuse into the reaction medium.
• the biomass is preferably cooled to a temperature below 40 ° C, or even chilled around 4 ° C.
• the applicant company considers that the heat treatment, performed under these operating conditions, could thus act as a membrane embrittlement process that allows the spontaneous release of soluble components of the intracellular compartment, or even the extracellular matrix.
• organic substances such as carbohydrates (predominantly DP1 and DP2), peptides and polypeptides are drained out of the cell.
• the process according to the invention therefore does not lead to the formation of an emulsion, but to an aqueous suspension.
• a lag time may be necessary to allow sufficient diffusion after the heat treatment which permeabilizes the membrane.
• the permeabilized biomass and the soluble fraction are then separated by a centrifugation technique, more particularly multi-stage centrifugation.
• the soluble fraction thus obtained may be clarified by microfiltration so as to rid it of the residual insolubles and according to its dry matter, a concentration by evaporation or by any means known to those skilled in the art may be carried out before purification which follows.
• the resulting soluble fraction is ultimately composed essentially of proteins (50-80% w / w) and carbohydrates (5-25% w / w).
• the residual biomass from which the solubles have been separated can be valued as a complete ingredient with a rebalanced nutritional profile.
• the protein content is decreased - because partly driven in the form of peptides in the soluble - and this rebalances the balance in favor of the carbohydrate and lipid fraction.
• the residual biomass after separation by centrifugation can also be milled (depending on the desired application properties), preferably mechanical grinding.
• the biomass is stabilized (pH readjusted (around 7), addition of antioxidants 7), it is then heat treated (pasteurization for bacteriological control purposes) before spray drying.
• a concentration step by evaporation can precede the heat treatment (optimization coupled with drying).
• the method of the invention leads here to the isolation of peptides of interest, by precipitation by modulating the properties of the medium.
• the cooling temperature is below 10 ° C, preferably below 4 ° C.
• the pH in addition to the temperature, the pH must be between 2.5 and 6.5 and preferably be close to pHi, ie between 3 and 5.
• the ionic strength of the medium can be adapted to promote precipitation.
• the phenomenon of "salting in” can be attenuated and thus the solubility of the proteins can be reduced (by reducing the solvation layer).
• a demineralization operation prior to precipitation can be added. This is carried out on cationic and anionic resins, dialysis, filtration or by any means known to those skilled in the art. Conversely, by increasing strongly the ionic strength, the available water decreases by the phenomenon of "Salting out", in this way the proteins tend to precipitate. This mode is not preferred since a pronounced demineralization would then be necessary on the protein isolate thus extracted.
• the polarity of the medium can be decreased (with dehydration of the medium) by adding an ethanol-type solvent which will generate a more quantitative precipitation of the protein fraction by greatly reducing its solubility. by recovering the precipitated fraction which is then optionally concentrated before drying
• the separation of the precipitated fraction is carried out by simple decantation and recovery of the heavy phase or optionally by centrifugation under optimum temperature conditions.
• the pH may be readjusted before drying.
• the drying is carried out by atomization, lyophilization or by any means known to those skilled in the art.
• the soluble phase (light phase after separation) can be valorized as such as a protein concentrate (according to its residual protein content) or undergo a purification process again to extract the residual peptides. .
• the residual peptides can be extracted by modulation of the physico-chemical environment in the same manner as described for the protein isolate.
• the incorporation of an ethanol-type solvent can be carried out at this stage to generate a precipitation of this residual protein fraction by greatly reducing its solubility.
• the action of the solvent will be all the more effective if the residue is dehydrated beforehand. This can be done up to a certain dry matter by evaporation or until a complete drying (for example, by atomization).
• the pH of this fraction may optionally be readjusted then, a concentration by evaporation (which may allow the recycling of the solvent) is optionally performed before spray drying, lyophilization or by any means known to those skilled in the art.
• strain CCAP21 1 / 8D The Culture Collection ofAlgae and Protozoa, Scotland, UK.
• Composition of the medium 40 g / l of glucose + 10 g / l of yeast extract.
• diammonium phosphate 20 g / l
• magnesium sulfate heptahydrate 12 g / l
• This fermentation line makes it possible to obtain a biomass having more than 65% of proteins expressed in N, 6.25.
• the biomass produced according to Example 1 is harvested at a dry cell solids content of 105 g / L with a purity of 80% (purity defined by the ratio between the dry matter of the biomass on total dry matter).
• the heat treatment is carried out at a moderate scale to limit the partial solubilization of the biomass which sees its purity decrease to 68%.
• composition of the biomass is as follows:
• the separation of the solubles resulting from the release by thermal permeabilization of biomass is carried out by centrifugal separation.
• a slight dilution [0.5: 1] (VeauVmout) is performed online on the second stage (on a configuration with two Alfa Laval FEUX 510 centrifuges in series) with recycling. from the supernatant of the second stage to the first. The supernatant of the first stage is thus recovered and concentrates the clarified solubles.
• a sample of solubles taken after separation is used for purification to obtain the protein isolate.
• the pH of the crude solubles is adjusted to 4.5 with phosphoric acid.
• the heavy phase is then extracted by simple phase separation into a separating funnel with a mass yield of 28% and has a solids content of 37.2%.
• the distribution of the amino acid profile of the protein isolate is as follows:
• the isolate is therefore characterized by a richness of about 95% in amino acids consisting essentially of arginine and glutamic acid (based on the analysis of the distribution of total amino acids). Purification of the residue
• the light phase, after precipitation and separation of the isolate can be purified in order to concentrate the protein fraction having not precipitated (lower molecular weight).
• this phase is concentrated by evaporation (15mBar - 43 ° C. in the Buchi -215 laboratory rotavapor) to a solids content of 45%. , 4% in order to partially dehydrate the medium to then promote the action of ethanol.
• the concentrate has the following composition:
• the pellet is recovered by centrifugation at 4000 g for 10 minutes (Beckman Coulter Avanti J-20 XP).
• This extract can then be promoted as a protein concentrate.
• Example 2 The crude solubles obtained in Example 2, which are rich in proteins, are separated from the residual biomass that can be treated by a process that allows its recovery.
• the biomass extracted to a dry cell solids content of 22% is milled on a Bead Mill horizontal ball mill (NETZSCH LME 500 - 0.6 mm zirconium silicate beads) at a milling rate of 85%.
• NETZSCH LME 500 - 0.6 mm zirconium silicate beads The biomass extracted to a dry cell solids content of 22% is milled on a Bead Mill horizontal ball mill (NETZSCH LME 500 - 0.6 mm zirconium silicate beads) at a milling rate of 85%.
• the pH of the cell grind is then adjusted to 7 at 50% potash.
• Concentration on forced flow evaporator SPX is carried out by continuous feed of a loop where the temperature is adjusted to 75 ° C before the entry of the flash under vacuum with the temperature maintained at 40 ° C where the evaporation takes place.
• the concentrated biomass is continuously withdrawn from the flash to the UHT SPX module to perform a heat treatment with preheating at 70 ° C and then direct steam injection on a scale of about ten seconds at 140 ° C and cooling at 40 ° C by flash at empty.
• the biomass is then atomized to a dry matter of 95% on a type GEA Filtermat FMD 200 atomizer.
• the biomass thus obtained has the following composition:
• the biomass thus obtained has the advantage of having a balanced nutritional profile at the level of the carbohydrate, protein and lipid fraction.
• the amino acid profile is, moreover, rebalanced by the elimination upstream of the soluble fraction that is selectively rich in arginine and glutamic acid.
• the distribution of amino acids in biomass is as follows:

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• 2015
  • 2015-01-26 FR FR1550571A patent/FR3031987B1/fr active Active
• 2016
  • 2016-01-25 BR BR112017014583A patent/BR112017014583A8/pt not_active Application Discontinuation
  • 2016-01-25 CN CN201680007322.6A patent/CN107208124A/zh active Pending
  • 2016-01-25 JP JP2017539262A patent/JP2018502592A/ja active Pending
  • 2016-01-25 WO PCT/FR2016/050138 patent/WO2016120548A1/fr active Application Filing
  • 2016-01-25 EP EP16705227.3A patent/EP3250704A1/de not_active Withdrawn
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  • 2016-01-25 US US15/546,236 patent/US20180007932A1/en not_active Abandoned
  • 2016-01-25 KR KR1020177017771A patent/KR20170105497A/ko unknown

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