EP3250705A1 - Method for obtaining a peptide isolate from a biomass of protein-enriched microalgae - Google Patents

Abstract

The invention relates to a peptide isolate isolated from a biomass of protein-enriched microalgae. The invention is characterized in that it comprises: - soluble peptides with a molecular weight between 1 and 20 k Da, - a protein content expressed in N 6.25 of more than 95%, - essentially arginine and glutamic acid.

Description

 PROCESS FOR OBTAINING PEPTIDE ISOLATE FROM BIOMASS OF MICROALGUES ENRICHED IN PROTEINS

The present invention relates to a peptide isolate derived from a biomass of microalgae rich in proteins, microalgae of the genus Chlorella, more particularly of the species Chlorella protothecoides.

The algae, macro and micro, have a richness largely unexplored. Their exploitation for food, chemical or bioenergetic purposes is still very marginal. However, they contain components of great value, wealth and abundance.

 Microalgae are indeed sources of vitamins, lipids, proteins, sugars, pigments and antioxidants.

 Algae and microalgae are thus of interest to the industrial sector that uses them for the manufacture of food supplements, functional foods, cosmetics, medicines or for aquaculture.

 Microalgae are primarily photosynthetic microorganisms that colonize all biotopes exposed to light.

On an industrial scale, their monoclonal culture is carried out in photobioreactors (autotrophic conditions: in the light with C0 ₂ ) or for some of them, also in fermenters (heterotrophic conditions: in the dark in the presence of a carbon source).

 Some species of microalgae are indeed able to grow in the absence of light: Chlorella, Nitzschia, Cyclotella, Tetraselmis, Crypthecodinium, Schizochytrium.

 It is moreover estimated that culture in heterotrophic conditions costs 10 times less expensive than in phototrophic conditions because, for the skilled person, these heterotrophic conditions allow:

 - The use of fermenters identical to those used for bacteria and yeasts and allow the control of all culture parameters.

 - the production of biomasses in a quantity much higher than what is obtained by culture based on the light.

 The profitable exploitation of the microalgae generally requires the control of the conditions of fermentation making it possible to accumulate its components of interest, such as:

the pigments (chlorophyll a, b and c, β-carotene, astaxanthin, lutein, phycocyanin, xanthophylls, phycoerythrin, etc.) whose demand is increasing, both for their remarkable antioxidant properties and for their contribution of natural colors in the 'food, lipids, in order to optimize their fatty acid content (up to 60% or even 80% by weight of their dry matter), in particular for:

 • biofuel applications, but also

 • Food or feed applications, when the selected microalgae produce polyunsaturated fatty acids or

PUFAs called "essential" (i.e. brought by the diet because are not naturally produced by the man or the animal), or

 proteins, in order to optimize their nutritional qualities or, for example, to promote the supply of amino acids of interest by means of the preparation of peptide-rich fractions.

Peptide fractions can be promoted as functional agents or dietary supplements in many fields.

 In the context of the amino acid supply of interest, it may indeed be advantageous to have sources of peptides rich in arginine and glutamic acid.

 Arginine is an amino acid that has many functions in the animal kingdom.

 Arginine can be degraded and thus serve as a source of energy, carbon and nitrogen to the cell that assimilates it.

 In various animals, including mammals, arginine is decomposed into ornithine and urea. The latter is a nitrogen molecule that can be eliminated (by excretion in the urine) so as to regulate the amount of nitrogen compounds present in the cells of animal organisms.

 Arginine is used to synthesize nitric oxide (NO) with NO synthase, thereby mediating arterial vasodilatation, which reduces the rigidity of blood vessels, increases blood flow, and improves the functioning of blood vessels.

 Dietary supplements that contain arginine are recommended to promote heart health, vascular function, to prevent "platelet aggregation" (risk of blood clots) and to lower blood pressure.

 The involvement of arginine in wound healing is related to its role in the formation of proline, another important amino acid for collagen synthesis.

 Arginine is also a component frequently used, especially by athletes, in energy drinks.

Glutamic acid is not only one of the building blocks used for protein synthesis, but it is also the excitatory neurotransmitter more prevalent in the central nervous system (brain + spinal cord) and is a precursor of GABA in GABAergic neurons.

 Under the code of "E620", glutamate is used as a food flavor enhancer. It is added to food preparations to enhance their taste.

 In addition to glutamate, the Codex Alimentarius has also recognized as flavor enhancers its salts of sodium (E621), potassium (E622), calcium (E623), ammonium (E624) and magnesium (E625).

 Glutamate (or its salts) is often present in ready-made dishes (soups, sauces, chips, ready meals). It is also commonly used in Asian cooking.

 It is currently frequently used in combination with aromas in appetizers (bacon taste, cheese taste). This enhances the taste of bacon, cheese, etc. It is rare to find an aperitif that does not contain any.

 It is also found in some drug capsules but not for its taste functions.

 Finally, it is the majority component of kitchen auxiliaries (cubic broths, sauces, sauces, etc.).

However, developments in food applications of peptide fractions of microalgae have not been significant because of the presence in said fractions of undesirable compounds (pigments ...). These compounds lead to undesired changes in the color, taste and structure of the food compositions containing them.

 To increase their potentiality in food applications and also to increase their commercial value, these peptides must be extracted from the microalgae with the required compositions, in terms of:

 - nitrogen richness,

 - richness in amino acids of interest,

 - low pigment content Object of the invention

 The present invention relates to a peptide isolate derived from a biomass of microalgae rich in proteins, microalgae of the genus Chlorella, more particularly of the species Chlorella protothecoides.

More particularly, the present invention relates to a microalgae isolate characterized by its remarkably high content of arginine and glutamic acid. The present invention also relates to the biomass of microalgae rich in proteins as such, biomass particularly suitable for the preparation of said peptide isolate.

 The present invention also relates to the process for enriching and depigmenting the biomass of microalgae, more particularly of the genus Chlorella, more particularly of the species Chlorella protothecoides.

 The present invention finally relates to the process for preparing this peptide isolate from the biomass of microalgae rich in proteins and depigmented.

 Indeed, in order to exploit the metabolic wealth of microalgae, and more particularly their peptide fractions, the applicant company proposes to provide a peptide isolate having:

 soluble peptides with a molecular weight of between 1 and 20 kDa,

- a protein content expressed in N 6.25 of more than 95%,

 essentially arginine and glutamic acid.

 Within the meaning of the invention, the term "essentially composed of arginine and glutamic acid" means a richness in arginine and glutamic acid that can be understood as an arginine and glutamic acid content of more than 80, 85, 90 or 95% by weight expressed relative to the total amino acids. In particular, these two amino acids represent 85 to 99% relative to the total amino acids, preferably between 90 and 98% relative to the total amino acids, and in particular between 95 and 98%.

 More specifically, within the meaning of the invention, the term "essentially composed of arginine and glutamic acid" means a richness in arginine and glutamic acid that can be understood as containing:

 at least 40% arginine, in particular between 40% and 60%, preferably around 50%; and

 at least 40% glutamic acid, in particular between 40% and 60%, preferably about 50%

 expressed on total amino acids.

 In particular, the amino acid content other than arginine and glutamic acid is less than 10%, preferably less than 5%, especially less than 3%.

 In a specific embodiment, the content of the isolate is as follows:

a content of between 47 and 48% of arginine;

 a content of between 49 and 50% of glutamic acid;

expressed on total amino acids, which results in an amino acid content other than arginine and glutamic acid of less than 3%. By "about" is meant a range of value comprising + or - 10% of the indicated value, preferably + or - 5% thereof. For example, about 10 is between 9 and 11, preferably between 9.5 and 10.5.

 This isolate of peptides can be prepared from a biomass of microalgae of the genus Chlorella, more particularly of the species Chlorella protothecoides, microalgae discolored and having a protein content, expressed in N 6.25 greater than 60%, for example more than 65%.

 The preferred method of microalgae fermentation is a two-step process comprising:

a first fermentation stage, deficient in nitrogen, where the regulation of the pH is carried out by a mixture ΝΗ ₃ / ΚΟΗ then,

a second step of removing this nitrogen deficiency by pH regulation provided by NH ₃ alone.

 These operating conditions thus make it possible to rapidly obtain a biomass having a protein content of greater than 60% in N 6,25, of the order of or around 65% in N 6,25, and a weak coloration. The yield is 45 to 50% by weight of dry matter, and the final concentration of biomass is between 80 and 120 g / l.

 In addition, the residual salt content of the soluble fraction of the fermentation must does not exceed 6 g / l.

 The biomass thus prepared is then washed in order to purify it from its interstitial solubles (in particular soluble salts), brought to a solids content of between 15 and 30%, preferably to a solids content of between 20 and 30%, and then is heat-treated. at a temperature between 50 and 150 ° C, for a period of between 5 seconds and 5 minutes.

 At the end of this treatment, which permeabilizes the cell membrane and allows the release of the soluble components of the intracellular compartment by free diffusion, the residual biomass is removed, and the recovered soluble fraction is then clarified, precipitated, concentrated and then dried to form the peptide isolate according to the invention.

 The present invention thus relates to an isolate obtained or obtainable from a protein rich microalgae biomass prepared by a fermentative method described herein. It also relates to an isolate obtained or obtainable from the biomass of high protein microalgae by a method of treating biomass as described herein. Detailed description of the invention

Characterization of the peptide isolate according to the invention The invention relates to a peptide isolate prepared from a biomass of microalgae grown so as to enrich it with proteins, microalgae of the genus Chlorella, more particularly Chlorella protothecoides.

 The peptide isolate according to the invention, obtained from this protein-rich biomass, is characterized in that it comprises:

 soluble peptides with a molecular weight of between 1 and 20 kDa,

a protein content expressed in N 6.25 of more than 95%,

 - essentially arginine and glutamic acid.

 In this context of defining the isolate, the term "comprises" means that the isolate consists essentially of these peptides but may comprise other minority components. Thus, "essentially constituted" means at least 90, 95 or 99% by dry weight of the isolate.

 The molecular weight of said peptides is measured by chromatography according to the following method:

 Chromatographic conditions:

 - 2 columns mounted in series: SUPERDEX® 200HR10 / 30 column with a SUPERDEX® Peptide HR10 / 30 column (from Pharmacia Biotech)

- Flow rate: 0.3 ml / min

 UV detector at 214 nm

 - Eluent: 0.05 M NaCl

 Analysis time: 240 min

 The sample is dissolved in 0.5% water in HPLC grade water.

 The columns are calibrated with a control mixture Biorad ref 151 -1901 composed of:

 Thyroglubulin: Mw = 670 KDa

 - Bovine globulin: Mw = 158 KDa

 Ovalbumin: Mw = 44 KDa

 Myoglobin: Mw = 17 KDa

 - Vitamin B12: Mw = 1 .35 KDa

 The% of the different fractions is then calculated on the basis of the retention times of each control.

 The measurement of the protein content is conventionally determined by the measurement of N.6.25, known in general

 Finally, the amino acid composition is determined according to NF EN ISO 13903 (November 2005).

The arginine and glutamic acid contents of the isolate are as specified hereinabove. The high content of arginine and glutamic acid is understood here for example by a content:

 - between 47 and 48% arginine

 - between 49 to 50% of glutamic acid

 expressed on total amino acids, which results in an amino acid content other than arginine and glutamic acid of less than 3%.

 Optionally, the peptide isolate comprises less than 3% of total sugars (carbohydrates). Process for preparing the peptide isolate according to the invention

 The peptide isolate according to the invention can be prepared from a biomass of microalgae of the genus Chlorella, more particularly of the species Chlorella protothecoides, microalgae discolored and having a protein content, expressed in N.6, 25 greater than 60%.

 To obtain maximum yields and productivities in proteins, the applicant company has implemented an original process that it has also protected in one of its recently filed patent applications.

 In the state of the art, early fermentation processes to obtain high cell densities (HCD for High-Cell-Density) had been much worked.

 The objective of these HCD cultures was to obtain the highest possible concentration of the desired product in the shortest time.

However, maintaining growth at its maximum rate (μ, in h ¹ ) is not always correlated with the high production of the product of interest.

 Therefore, in cases where the formation of the products is not correlated with high or maximum cell growth, it is advisable to control the rate of cell growth.

 In general, a person skilled in the art chooses to control the growth of microalgae by controlling the fermentation conditions (Tp, pH, etc.), or by the regulated feeding into nutritional components (nitrogen or carbon sources) of the fermentation medium. , under semi-continuous conditions called "fed-batch".

 Chlorella protothecoides is justly recognized as one of the best oil-producing microalgae.

In heterotrophic conditions, it rapidly converts carbohydrates into triglycerides (more than 50% of its dry matter). To optimize this production of triglycerides, those skilled in the art are led to optimize the carbon flow towards the production of oil, by acting on the nutritional environment of the fermentation medium.

 It is thus known that the accumulation of oil occurs during a sufficient carbon supply, but under conditions of nitrogen deficiency.

 The C / N ratio is here decisive, and it is accepted that the best results are obtained by acting directly on the nitrogen content, the glucose content not being limiting.

 But Chlorella protothecoides can also be used for its ability to produce proteins.

 For the production of protein-rich biomasses, a person skilled in the art is therefore led to take the opposite of the metabolic control allowing the microalgae to naturally produce reserve lipids, that is to say to work the fermentation conditions by favoring a low C / N ratio, and thus:

 - make a significant contribution of nitrogen source to the fermentation medium, while maintaining constant the carbon source feed that will be converted into proteins, and

 stimulate the growth of the microalgae.

 It is a question of modifying the carbon flow towards the production of proteins (and thus of biomasses), to the detriment of the production of the reserve lipids.

 In the context of the invention, the applicant company has instead chosen to explore an original way by proposing alternative solutions to those conventionally envisaged by those skilled in the art.

 The heterotrophic culture process of said microalgae developed by the Applicant Company to increase the protein content of the biomass then comprises:

 a so-called "batch" fermentation phase characterized, after the fermenter has been inoculated, with the addition of a quantity of glucose of between 15 and 25 g / l, preferably of the order of 20 g / l, in one go;

an exponential fed batch phase during which the glucose is brought gradually and the pH regulation initially made by the NH 3 / KOH mixture is replaced by regulation with NH ₃ alone.

As will be exemplified below, the NH ₃ contribution remarkably rapidly induces an increase in the level of proteins synthesized in the cell, which results in an increase in the intracellular N 6,25 level to a value exceeding the 60%. A complete analysis of the amino acids present in the biomass was then performed on a sample taken just before the change of pH regulation, and on several other samples taken after said change.

 It is observed that before the change, the sum of the amino acids is low (of the order of 15 to 25%) and that there is no predominance among the different amino acids.

 After the change of regulation, we note that:

 the sum of the amino acids exceeds 40%,

 in total, the content of glutamic acid and arginine on total amino acids is more than 45%,

 the amino acid which knows the strongest increase is glutamic acid, followed by arginine. The rate of other amines also increases, but much less.

 The increase in N.6, 25 is therefore directly correlated with the increase in the synthesis of glutamic acid and arginine.

 Moreover, this mode of pH regulation makes it possible:

 to limit the salt intake of the fermentation medium, and

 - to influence the color of the biomass, which will be even more yellow when the NH3 content has initially been limited.

 In conclusion, the biomass of microalgae rich in proteins, microalgae of the genus Chlorella, more particularly of the species Chlorella protothecoides presents:

 a concentration of 80 to 90 g / l,

 - a N.6,25 content of more than 60%,

 a quantity of salts of less than 6 g / l in its culture supernatant,

a weak coloration, and

 an amino acid and glutamine content of more than 45% by weight on total amino acids.

 This biomass is particularly well suited to the preparation of the peptic isolate according to the invention, by implementing the following method:

 optionally, washing the biomass so as to eliminate the interstitial soluble compounds,

thermal permeabilization of the biomass 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 10 seconds and approximately 1 minute, elimination of the biomass thus permeabilized by a solid-liquid separation technique, preferably chosen from the group consisting of frontal or tangential filtration, flocculation and centrifugation, more particularly multi-stage centrifugation, to obtain a soluble fraction,

 optionally, recovery and clarification of the soluble fraction thus obtained by microfiltration so as to rid it of residual insolubles,

 purification of the soluble fraction by precipitation, in order to obtain a peptide isolate, and

 evaporation, pasteurization and atomization of said peptide isolate.

 After fermentation under the conditions set out above, the biomass is collected by solid-liquid separation, by frontal or tangential filtration or by any means known to those skilled in the art.

 Optionally, the Applicant Company recommends washing the biomass so as to eliminate the interstitial soluble compounds by a succession of concentration (by centrifugation) / dilution of the biomass.

 For the purposes of the invention, the term "interstitial soluble compounds" means all the soluble organic contaminants of the fermentation medium, for example the water-soluble compounds such as soluble 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 about 80 and 150 ° C, for a period of time between about

5 seconds and about 5 minutes, preferably for a period of time

10 seconds and about 1 minute. Preferably, the heat treatment is carried out at a temperature of about 140 ° C for a period of about 10 seconds. In another preferred alternative, the heat treatment is carried out at a temperature of about 85 ° C for a period of about 1 minute.

 This treatment makes it possible to allow the intracellular components to diffuse into the reaction medium.

 Finally, at the end of these steps, the biomass is cooled to a temperature below 40 ° C, preferably cooled to a temperature of the order of 4 ° C.

Without being bound by any theory, the applicant company considers that the heat treatment, carried out under these operating conditions, could act thus as a membrane embrittlement process that allows the spontaneous release of soluble components of the intracellular compartment, or even the extracellular matrix.

 In addition to ionic substances, organic substances such as carbohydrates (predominantly DP1 and DP2), peptides and polypeptides are drained out of the cell.

 On the contrary, the lipids and hydrophobic organic compounds remain in the cells, which clearly demonstrates that the cells are permeabilized and not lysed or destroyed.

 The process according to the invention therefore does not lead to the formation of an emulsion, but to an aqueous suspension.

 The release of all these soluble substances through the permeabilized membrane is similar to a dialysis free diffusion method.

 As a consequence, a lag time may be necessary to allow sufficient diffusion after the heat treatment which permeabilizes the membrane.

 In the literature, the pulsed field permeabilization method of yeast membranes to extract the proteins takes 4 to 6 hours (Ganeva et al., 2003, Analytical Biochemistry, 315, 77-84).

 According to the invention, a much shorter reaction time is implemented, between 5 seconds and 5 minutes.

 The rise in the scale (time / temperature) then leads to an increase in the solubilization rate and the extraction yield of the solubles.

 The method of the invention advantageously exploits the thermal permeabilization phenomenon to extract the solubilized peptide fraction of the residual biomass.

 Thus, the residual biomass is then removed by a solid-liquid separation technique by frontal or tangential filtration, by flocculation, by centrifugation, or by any other means known to those skilled in the art, which makes it easy to recover the fraction. soluble free of microalgae cells.

 The yield and quality of this separation step can be improved by diluting the permeabilized cells (for example by multi-stage dilution / centrifugation).

If necessary, 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 (10-25% w / w).

 The conventional processes for recovering soluble proteins generally rely on a step of precipitating said proteins with trichloroacetic acid (10% w / v) or with ammonium sulphate.

 However, these precipitation isolations follow very destructive cell breaking processes (most often by sonication or homogenization) which, if they make it possible to increase the extraction yields, lead mainly to low solubility proteins. and denatured.

 Their refunctionalization can then be considered only through their hydrolysis product (in peptides) by chemical means (lysis with soda), physical (high temperature treatment) or enzymatic (proteolytic enzymes).

 The method of the invention then leads to the isolation of the proteins of interest, by precipitation by modulating the properties of the medium.

 The applicant company therefore recommends that:

 to promote the precipitation of all or part of the protein fraction by modulating the physicochemical properties of the medium.

 Cooling of the crude solubles, obtained as described in the preceding steps, triggers a precipitation phenomenon of a part of the soluble peptides. It is observed that the precipitation is rather selective on the higher molecular weights. The cooling temperature is below 10 ° C, preferably below 4 ° C.

 Certain operating conditions make it possible to promote this phenomenon: 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.

 In the same way, the ionic strength of the medium can be adapted to promote precipitation. Thus, by greatly reducing the ionic strength, it is possible to reduce the phenomenon of salts ("Salting-in") and thus reduce the solubility of the proteins (by reducing the solvation layer). Thus, 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 strongly increasing 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.

 In the same optics of modulation of the solvation layer, 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.

 Prior to drying, the incorporation of a concentration step by evaporation can make it possible to optimize the operation energetically. It can be especially justified if a solvent of the ethanol type is used to allow its recycling.

 The exploitation of these channels makes it possible to purify a fraction with a high content of peptides and polypeptides of salts and residual sugars.

 A soluble protein isolate greater than 90% by weight, rich in arginine and glutamic acid, is then obtained.

The invention will be better understood with the aid of the examples which follow, which are intended to be illustrative and not limiting.

Examples

Example 1: Preparation of a biomass of C. protothecoides rich in proteins with a high content of glutamic acid and arginine

The strain used is Chlorella protothecoides (strain CCAP21 1 / 8D - The Culture Collection of Algae and Protozoa, Scotland, UK).

 Preculture:

 150 ml of medium in a 500 ml Erlenmeyer flask;

 - Composition of the medium: 40 g / l of glucose + 10 g / l of yeast extract.

 Incubation takes place under the following conditions:

- duration: 72 h; temperature: 28 ° C .;

 stirring: 10 rpm (Infors Multitron Incubator).

 Culture in batch mode then fed batch

 Preparation and initial batch medium

 preparing and filtering a KOH mixture at 400 g / l (41%) / NH3 at 20% v / v (59%);

- sterilize 20 L fermenter at 121 ° C / 20 min;

 inoculate with 2 500 ml preculture Erlenmeyer flasks (OD 600 nm of 15);

setting to pH 4.5 with NH ₃ 20% v / v;

 - agitation of 300 rpm departure;

 aeration: 20 L / min of air;

- regulation p0 ₂ to 30%;

 Food

 - glucose: 500 g / l

 - ammonium sulphate: 5 g / l

 - diammonium phosphate: 20 g / l

 phosphoric acid: 16 g / l

 - magnesium sulphate heptahydrate: 12 g / l

 - iron sulfate: 170 mg / l

 - calcium nitrate: 610 mg / l

 trace elements solution: 45 ml / l

 vitamin solution: 4.5 ml / l

 It is important to note that the loading of ammonium salts, magnesium salts and phosphoric acid was designed to limit the salt content of the fermentation medium and was optimized to maintain the N.6 content, 25 of the final discolored biomass.

Calcium Panthothenate 0.4

 P-aminobenzoic acid 0.8

Conduct of fermentation

 - bring the equivalent of 20 g / 1 before inoculation

- when the glucose concentration is = 0 g / 1, start the exponential profile feed (μ = 0.07 h ¹ );

- pH regulation at 5.2 with the mixture 41% KOH / 59% NH ₃

- when 2 kg of glucose are consumed by the microalgae, we switch to pH regulation by NH ₃ alone

 Results:

 This fermentation line makes it possible to obtain a biomass having more than 65% of proteins expressed in N, 6.25.

Example 2 Process for Thermal Permeabilization of Protein-enriched Biomass and Recovery of "Raw" Solubles

The biomass obtained 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).

 She is then:

- washed and concentrated by online dilution [1: 1] (V _Others uVmoût)

 - centrifuged on an Alfa Laval FEUX 510 plate centrifuge and brought to a solids content of 220 g / l and a purity of 93% (purity defined by the ratio of the dry matter of the biomass to the total dry matter).

 The pH is adjusted to 7 with potassium hydroxide and the biomass is thermally treated with UHT 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 vacuum flotation.

 The heat treatment is pushed to a high scale in order to maximize the partial solubilization of the biomass which sees its purity decrease to 53%.

 By definition, the release of solubles in the extracellular medium leads to a decrease in the cell dry matter fraction relative to the total dry matter.

 At this stage, the composition of the biomass is as follows:

 - Total amino acids: 48.4%

 - Total sugars: 27,2%

 - Total fatty acids: 15.1%

 - Ashes: 2.5%

- Other residues: 6.8%. The separation of the solubles resulting from the release by thermal permeabilization of biomass is carried out by centrifugal separation.

 In order to optimize the efficiency and the quality of the 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.

 These "raw" solubles have the following composition:

 - Total amino acids: 77.3%

 - Total sugars: 17.6%

 - Ashes: 4.3%

 - Other residues: 0.8%.

Example 3 Purification and Obtaining the Protein Isolate

In order to selectively precipitate the protein fraction, 5 kg of crude solids with a solids content of 11.4% are placed in a double jacket reactor with stirring.

 The pH of the crude solubles is adjusted to 4.5 with phosphoric acid.

 After stopping the stirring, the temperature is lowered to 4 ° C.

 These conditions are maintained for 8 hours.

 Thus the decantation of the heavy phase enriched in peptides of higher molecular weight takes place.

 The heavy phase is then extracted by simple phase separation into a separating funnel with a mass yield of 26% and has a solids content of 36.3%.

 This extract is lyophilized to a dry matter content of 97%.

 The composition of this isolate is as follows:

 - total amino acids: 95, 9%

 - total sugars: 2.44%

 - ash: 1, 66%

 The analysis of the amino acid distribution in the total amino acids is as follows:

 - glutamic acid: 49.78%

 - arginine: 47.21%

- other amino acids: 3,01% The isolate is characterized by a richness of the order of 95% in amino acids consisting essentially of arginine and glutamic acid (based on the analysis of the distribution of total amino acids).

 The molecular weight of this fraction is essentially between 1 kDa and 20 kDa.

Claims

1. A peptide isolate isolated from a biomass of protein-rich microalgae, characterized in that it has:

 soluble peptides with a molecular weight between 1 and 20 kDa, a protein content expressed in N.6,25 of more than 95%, mainly arginine and glutamic acid.

2. Isolate according to claim 1, characterized in that the microalgae are microalgae of the genus Chlorella, more particularly Chlorella protothecoides.

3. Isolate according to either of claims 1 and 2, characterized in that the content of arginine and glutamic acid is more than 80, 85, 90 or 95% by weight expressed relative to the total amino acids.

4. Isolate according to any one of claims 1 to 3, characterized in that the content of arginine and glutamic acid is

 at least 40% arginine, in particular between 40% and 60%, preferably about 50%; and

 at least 40% of glutamic acid, in particular between 40% and 60%, preferably about 50%

 expressed on total amino acids.

5. Isolate according to any one of claims 1 to 4, characterized in that the content of arginine and glutamic acid is:

 - between 47 and 48% for arginine

 - between 49 to 50% for glutamic acid

 expressed on total amino acids.

 6. Isolate according to any one of claims 1 to 5, characterized in that it is obtained or capable of being obtained from a biomass of microalgae rich in proteins prepared by a fermentation process which comprises:

 a so-called "batch" fermentation phase characterized, after the fermenter has been inoculated, with the addition of a quantity of glucose of between 15 and 25 g / l, preferably of the order of 20 g / l, in one go;

an exponential fed batch phase during which the glucose is brought gradually and the pH regulation initially made by the NH 3 / KOH mixture is replaced by regulation with NH ₃ alone.

The isolate according to any one of claims 1 to 6, characterized in that it is obtained or obtainable from the biomass of microalgae rich in proteins by a process which comprises:

 optionally, washing the biomass so as to eliminate the interstitial soluble compounds,

 the thermal permeabilization of the biomass at a temperature of between 50 and 150 ° C., preferably approximately 80 and 150 ° C., for a duration of between approximately 10 seconds and approximately 5 minutes, preferably for a duration of between approximately 10 seconds and about 1 minute,

the elimination of the biomass thus permeabilized by a solid-liquid separation technique preferably chosen from the group consisting of frontal or tangential filtration, flocculation and centrifugation, more particularly multi-stage centrifugation, to obtain a soluble fraction; ,

 optionally, recovering and clarifying the soluble fraction thus obtained by microfiltration so as to rid it of residual insolubles,

 purification of the soluble fraction by precipitation, in order to obtain a peptide isolate, and

 evaporation, pasteurization and atomization of said protein isolate.