WO2024218300A1

WO2024218300A1 - Method for degrading glycogen present in a biomass of galdieria

Info

Publication number: WO2024218300A1
Application number: PCT/EP2024/060721
Authority: WO
Inventors: Olivier CAGNAC; Axel ATHANE; Matthieu COURBALAY
Original assignee: Fermentalg
Priority date: 2023-04-19
Filing date: 2024-04-19
Publication date: 2024-10-24
Also published as: FR3147934A1

Abstract

The present invention relates to a treatment method for the degradation of the glycogen present in a biomass of unicellular red algae (ARU), particularly of the genus Galdieria, and to the product thus obtained.

Description

PROCESS FOR DEGRADATION OF GLYCOGEN PRESENT IN GALDIERIA BIOMASS

FIELD OF THE INVENTION

The present invention relates to a treatment method for the degradation of glycogen from a biomass of unicellular red algae (URA), particularly of the genus Galdieria, and the aqueous product thus obtained.

STATE OF THE ART

Unicellular red algae (URA), or Rhodophytes, are characterized by the presence of pigments within their cells. In addition to chlorophyll and carotenoids, unicellular red algae produce phycobiliproteins. These natural pigments resulting from photosynthesis are divided into four types: allophycocyanins, C-phycocyanins, phycoerythrins and phycoerythrocyanins.

Phycocyanins have beneficial properties for human and animal health, and are now used in many fields such as the pharmaceutical, cosmetic and food industries.

Microalgae belonging to the class Cyanidiophyceae, and more precisely to the genera Cyanidioschyzon, Cyanidium and Galdieria, are particularly interesting for the production of phycocyanins. The production of unicellular red algae biomass is well known to those skilled in the art, in particular for the production of molecules of interest, in particular the production of proteins such as phycocyanins. Methods for the production and extraction of said phycocyanins are described in the literature (WO2017/093345, WO2019/228947, WO2018/178334, WO2020/161280).

However, microalgae of this class, especially of the genus Galdieria, are also known to have glycogen as a major reserve sugar. This glycogen is a polymer of a(1 — > 4) glucoses branched by a-(1 — > 6) bonds. The glycogen of microalgae of the genus Galdieria has the particularity of having a large proportion of these branches, namely approximately 7 to 18% of branched glucoses randomly distributed along the molecule. This molecular structure gives glycogen a globular conformation making it soluble in water (Martinez-Garcia et al., Int J Biol Macromol. (2016) 89:12-8). In addition, if microalgae of the genus Galdieria possess the enzymes for the synthesis of highly branched glycogen, they also possess the enzymes to degrade it (Martinez-Garcia et al., Int J Biol Macromol. (2016) 89:12-18). These enzymes are intracellular enzymes. However, the intracellular medium of microalgae of this class has a pH between 6.3 and 7.1 (Miyagishima et al., Plant Cell Physiol. 62(6): 926-941 (2021)). Thus, these enzymes are known to have activity in this precise pH range.

For the preparation of aqueous extracts of microalgae, such as phycocyanin extracts of Galdieria sulphuraria, filtration steps may be necessary. Since glycogen is easily soluble in cold water, it is found in the aqueous fraction with the hydrophilic compounds of interest, such as phycocyanins. The filters used for the purification of phycocyanins retain all or part of the glycogen, and thus increase the viscosity of the retentate. This creates technical constraints in the process, such as problems with pressure build-up, flow reduction and clogging, particularly with tangential filtration membranes. Furthermore, if the glycogen is not removed, the prepared extract contains a lot of glycogen, is then viscous and has a low concentration of phycocyanins.

Glycogen is known to be resistant to certain enzymes. However, a process involving the addition of exogenous enzymes suitable for its degradation has already been developed (W02020/144330).

If it cannot be completely eliminated and/or inactivated, an enzyme added for the preparation of a food product must be listed in its composition. Such a non-eliminable substance corresponds to an additive or a technological aid which may pose marketing and/or formulation difficulties.

Other methods for preparing aqueous extracts of unicellular red algae biomass, particularly of the genus Galdieria, have been described in the literature (Moon et al., Korean J. Chem. Engl. 2016, 31, 3, pages 490-495). However, these methods remain difficult to apply on an industrial scale since they require either the addition of large quantities of ammonium sulfate resulting in effluents rich in pollutants that are undesirable for ecological and economic reasons, or the use of very expensive instruments such as chromatography, or a sequence of steps that is difficult to implement on a larger scale than the laboratory. In addition, none of these methods can selectively eliminate glycogen present in the biomass or in aqueous extracts of microalgae.

There is therefore a need to provide a process for degrading glycogen. present in a biomass of unicellular red algae whose main reserve sugar is glycogen, in particular in a biomass of microalgae of the genus Galdieria, while overcoming the problems of the prior art, and in particular those stated above.

PRESENTATION OF THE INVENTION

According to a first aspect of the invention, the inventors have developed a method for treating a biomass of unicellular red algae (URA) of the genus Galdieria, said method for treating URA biomass comprising the steps of: a) harvesting the biomass by separating the culture medium to obtain a crude URA biomass, b) cell lysis of the crude biomass from step (a) to obtain a lysate, c) optionally diluting the lysate from step (b) to obtain a solubilizer, and d) separating the insolubles suspended in the lysate from step (b) or the solubilizer from step (c) to obtain a clarifier, the method comprising a step of degrading the glycogen by keeping the crude biomass and/or the lysate and/or the solubilizer at rest for at least 3 hours, during which the liquid medium comprising the glycogen is at an acidic pH.

Surprisingly, the degradation of glycogen by the endogenous enzymes of the microorganism is effective over a wide pH range not limited to the intracellular pH of said microorganism with an optimum for more acidic pHs, in particular between 2 and 5. Still surprisingly, the enzymatic degradation works inside the cell in a treated biomass such as a raw biomass, a thawed raw biomass or a dried raw biomass but also in a lysate and a solubilize. The present invention also relates to a product capable of being obtained by a method according to the invention.

Advantageously, the method according to the present invention makes it possible to reduce or even eliminate the addition of exogenous enzymes suitable for the degradation of ARU glycogen and therefore to reduce the addition of substances which must be qualified as additives and/or technological aids.

Also, the method according to the invention applies with conditions of duration, temperature and pH from the mildest to the most drastic and can thus adapt to the stability of the molecules of interest to be extracted. DESCRIPTION OF FIGURES

Figure 1 shows free glucose concentrations in Galdieria sulphuraria biomass lysates over time at pH 3.75 and pH 6, at 37 °C without the addition of exogenous enzymes (SE: No Enzyme) and with 1% exogenous enzymes in the lysate (E: Enzyme).

Figure 2 shows free glucose concentrations in Galdieria sulphuraria biomass lysates at room temperature (“amb”, i.e. 20°C) in the absence of exogenous enzymes and at different pHs as a function of time.

Figure 3 shows free glucose concentrations in Galdieria sulphuraria biomass lysates at pH 3.75 at different temperatures, in the presence or absence of exogenous enzymes, as a function of time (SE: No Enzyme; E: Enzyme).

Figure 4 shows free glucose concentrations in Galdieria sulphuraria biomass lysates at pH 3.75 at different temperatures, in the absence of exogenous enzymes, as a function of time.

Figure 5 shows free glucose concentrations as a function of time in a lysate and solubilized Galdieria sulphuraria biomass at pH 3.75 at a temperature of 20°C in the absence of exogenous enzymes, and in a lysate of Galdieria sulphuraria biomass at pH 3.75 in the presence of exogenous enzymes (SE: Without Enzyme; E: Enzyme).

Figure 6 shows the percentage of glycogen digestion as a function of time in a clarified biomass of Galdieria sulphuraria at pH 4 at a temperature of 20°C in the absence or presence of exogenous enzymes.

Figure 7 shows the percentage of glycogen digestion as a function of time in a lysate, solubilizer and clarifier of Galdieria sulphuraria biomass at pH 4 at a temperature of 20°C in the absence of exogenous enzymes (SE: Without Enzyme).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the context of the present invention, the term "biomass" designates a set of microalgae cells, preferably produced by fermentation in a biological reactor. Said biomass can be seen as a mass of single-celled organisms.

Biomass can undergo different treatments and be raw biomass, lysed biomass, thawed raw biomass and/or dried raw biomass.

It is understood in the context of the present application that the properties of the biomass correspond to the average of the properties of all the cells constituting said biomass, in other words, a lysed biomass is a biomass comprising at least 50% of lysed cells relative to the total number of cells and a raw biomass may comprise lysed cells due to the harvesting step without being considered as a lysed biomass as long as the number of lysed cells out of the number of non-lysed cells remains in the minority, i.e. less than 50%.

The expression “raw biomass” designates a biomass obtained after harvesting, i.e. after recovery of the fermentation must then separation of the cells from at least part of the culture medium, possibly thawed and/or dried.

The term “lysed biomass” or “lysate” refers to a microalgae biomass in which at least 50% of the cells are lysed, preferably at least 70%, more preferably in which at least 80%, 85%, 90%, 95%, up to 100% of the cells are lysed.

The expression “thawed raw biomass” refers to raw biomass that has been frozen, possibly for storage and/or transport reasons, then thawed to reach a temperature suitable for its treatment according to the invention, in particular a temperature suitable for the step of degradation of the glycogen of the biomass according to the invention.

According to the invention, the expression “dried raw biomass” designates a raw biomass of microalgae which has been dried according to methods known to those skilled in the art and whose water content relative to the total weight of the biomass is less than 10%, preferably less than 7%, more preferably between 5% and 1% of water. Among the known drying methods, mention may be made of natural air drying, spray drying, fluidized air bed drying, drying using a roller dryer and freeze-drying.

The term “solubilized” refers to a lysed biomass or lysate that has undergone a dilution step with an aqueous solution of neutral, acidic or basic pH.

The term “clarifiate” corresponds to an aqueous extract obtained after separation of insoluble substances suspended in a lysate or solubilizer.

The term "rest" refers to a stage during which there is no modification of the chemical properties of the preferably dried or frozen raw biomass, lysate and/or solubilized material, by addition or extraction of one or more components. Rest does not exclude the fact that the raw biomass, lysate, solubilized material and/or clarified material may be mixed, i.e. stirred, under non-destructive conditions which do not affect the chemical properties of the raw biomass, lysate and/or solubilized material.

The term “liquid medium comprising glycogen” describes the intracellular medium of the cells constituting the crude biomass and/or the liquid phase of the lysate, solubilize and/or clarified where appropriate.

The expression “biomass treatment” refers to any process applied to biomass, in particular to modify its physicochemical properties, extract molecules of interest and/or purify them.

Note that the numerical intervals given are intended to include all intermediate numbers (for example, an interval from 1 to 5 includes, among others, 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

Note that all numerical values given refer to the actual value given as well as approximations of that value estimated from the general convention that the last digit given corresponds to the precision of the measurement. In the absence of precise error limits, the maximum error for the last digit specified must be estimated according to the rounding convention.

Treatment method according to the invention

The method according to the invention is a method for treating a biomass of unicellular red algae (URA) of the genus Galdieria, said method for treating URA biomass comprising the steps of: a) harvesting the biomass by separating the culture medium to obtain a crude URA biomass, b) cell lysis of the crude biomass from step (a) to obtain a lysate, c) optionally, diluting the lysate from step (b) to obtain a solubilizer, and d) separating the insolubles suspended in the lysate from step (b) or the solubilizer from step (c) to obtain a clarifier, the method comprising a step of degrading the glycogen by keeping the crude biomass and/or the lysate and/or the solubilizer at rest for at least 3 hours, during which the liquid medium comprising the glycogen is at an acidic pH.

Ideally, throughout the process, the conditions of temperature, duration and pH are particularly suitable for not degrading the compounds of interest in the aqueous extract, for example phycocyanins, while promoting the degradation of glycogen during the resting stage.

According to one embodiment, the glycogen degradation step is carried out on the raw biomass. This embodiment then comprises, in order, the steps of: a) harvesting the biomass by separating the culture medium to obtain a raw ARU biomass, resting the raw biomass for at least 3 hours during which the liquid medium comprising the glycogen is at acidic pH, b) cell lysis of the raw biomass from step (a) to obtain a lysate, c) optionally, diluting the lysate from step (b) to obtain a solubilizer, d) separating the insolubles suspended in the lysate from step (b) or the solubilizer from step (c) to obtain a clarifier, and optionally, a step of adjusting the pH of the raw biomass.

In this embodiment, the glycogen is in the intracellular medium of the cells constituting the biomass, which liquid medium is at an acidic pH, and the raw biomass is preferably a dried raw biomass or a thawed raw biomass. In the following two embodiments, after cell lysis, the glycogen is found in the liquid phase of the lysate, then where appropriate of the solubilize.

According to a preferred embodiment, the glycogen degradation step is carried out on the lysate. This embodiment then comprises, in order, the steps of: a) harvesting the biomass by separating the culture medium to obtain a crude ARU biomass, b) cell lysis of the crude biomass from step (a) to obtain a lysate, resting the lysate for at least 3 hours during which the liquid medium comprising the glycogen is at an acidic pH, c) optionally, diluting the lysate from step (b) to obtain a solubilizer, d) separating the insolubles suspended in the lysate from step (b) or the solubilizer from step (c) to obtain a clarifier, and optionally, a step of adjusting the pH of the crude biomass or the lysate.

It may be necessary, as needed, to adjust the pH of the liquid medium of the raw biomass and/or the lysate to obtain the desired acidic pH for the resting step on the lysate. According to another embodiment, the glycogen degradation step is carried out on the solubilisate. This embodiment then comprises in order the steps of: a) harvesting the biomass by separation of the culture medium to obtain a crude ARU biomass, b) cell lysis of the crude biomass from step (a) to obtain a lysate, c) dilution of the lysate from step (b) to obtain a solubilisate, resting for at least 3 hours of the solubilisate during which the liquid medium comprising the glycogen is at acidic pH, d) separation of the insolubles suspended in the solubilisate from step (c) to obtain a clarifier, and optionally, a step of adjusting the pH of the crude biomass, the lysate or the solubilisate.

It may be necessary, as needed, to adjust the pH of the liquid medium of the raw biomass, the lysate and/or the solubilisate to obtain the desired acidic pH for the resting step on the solubilisate. a) Harvesting the biomass

The biomass according to the invention is a biomass of unicellular red algae (URA), more particularly a biomass of microalgae producing phycocyanins having a high glycogen content.

These microalgae belong to the class Cyanidiophyceae including the genera Galdieria, Cyanidium and Cyanidioschyzon. Preferably, the microalgae are of the genus Galdieria.

Among the microalgae of the Galdieria genus, mention may in particular be made of the species Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria, Galdieria phlegrea, Galdieria javensis, Galdieria yellowstonensis, Galdieria sp Preferably, the biomass according to the invention is a biomass of Galdieria sulphuraria .

The methods for producing biomass from unicellular red algae (URAs), and in particular from the genus Galdieria, are well known to those skilled in the art. In the context of the present invention, the culture of microalgae can be carried out by any known culture technique, in containers adapted to the growth of microorganisms also called biological reactors, bioreactors or fermenters.

According to the invention, the biomass of unicellular red algae is obtained from microalgae cultivated industrially in a large capacity reactor, preferably to obtain fermentation musts comprising high densities of microorganisms. In the context of the present application, "high densities" means an amount corresponding to more than 50 g of dry matter per liter of fermentation must, preferably more than 100 g per liter. Examples of single-cell red algae cultures are described in the patent applications (WO2017/050917, WO2017/050918, WO2017/093345 and WO2019/228947). It is understood that a person skilled in the art will be able to determine the optimal parameters and conditions for culturing the microorganisms, such as the temperature conditions, lighting, duration of culture or even the nature and quantity of nutrients to be provided. The method according to the invention comprises a step a) of harvesting the biomass.

After culturing the microorganisms and obtaining a biomass, it is harvested to obtain a raw biomass. The harvesting of unicellular red algae can be carried out by any technique known to those skilled in the art, in particular by filtration, possibly gravimetric or under reduced pressure, decantation, precipitation followed by gravimetric filtration or even centrifugation.

The method according to the invention comprises a step a) of harvesting the biomass corresponding therefore to the recovery of the fermentation must followed by the separation of the cells of the biomass from at least part of the culture medium.

This step allows the production of raw biomass. The raw biomass thus harvested can also undergo a washing step, preferably with water, in order to eliminate certain soluble impurities.

The raw biomass obtained after harvesting and optionally after one or more washes comprises at least 70% water, and up to 90% water, preferably it comprises 75 to 88% water.

Preferably, the raw biomass according to the invention has a dry matter content of 5 to 30% by weight relative to the total weight of the raw biomass, generally still preferentially from 10 to 25% by weight, more preferentially from 10 to 20% by weight.

Also preferably, the raw biomass according to the invention has a C-phycocyanin concentration of between 3 and 12%. b) Cell lysis

The method for treating a biomass of unicellular red algae according to the invention comprises a step b) of cell lysis of the raw biomass from step a) to obtain a lysate. Preferably, step b) of cell lysis is carried out on a raw biomass whose dry matter content is from 5 to 30% by weight relative to the total weight of the raw biomass, preferably from 10 to 25% by weight, more preferably from 10 to 20% by weight.

Also preferably, step b) of cell lysis is carried out on a raw biomass according to the invention with a C-phycocyanin concentration of between 3 and 12%.

Cell lysis can be carried out by any means of lysis known to those skilled in the art, in particular by enzymatic, mechanical and/or chemical means.

In the context of the invention, prior to this lysis step b), the raw biomass may have undergone a washing, freezing, thawing, drying and/or rehydration step. In other words, in the context of the present invention, the raw biomass may in particular be a thawed and/or dried biomass.

In the preferred case of mechanical lysis, among the mechanical means that can be used according to the invention, mention will be made in particular of ball mills, mixer-dispersers, high-pressure homogenizers, bag mills, impact mills, ultrasound, or pulsed electric fields. As devices for implementing these methods, reference will be made for the ball mill: Discus-100 from Netzsch or ECM-AP60 from WAB, for the high-pressure homogenizer: Ariete from GEA, for the mixer-disperser: 700-X from Silverson, for the spindle mill: Contraplex from Hosakawa and for the impact mill: Condux from Netzsch.

Preferably, cell lysis is done by mechanical lysis, preferably by grinding and even more preferably with a ball mill.

Preferably, the lysate obtained has a dry matter content of 5 to 30% by weight relative to the total weight of the lysate, preferably 10 to 25% by weight, more preferably 10 to 20% by weight.

Also preferably, the lysate obtained has a C-phycocyanin concentration of 3% to 12% relative to the total weight of the dry matter.

This lysis step b) can be carried out before or after the glycogen degradation step, i.e. before or after the resting step, preferably before. c) Optional dilution of the lysate

According to the invention, the lysed biomass or lysate can optionally undergo a dilution step. In the context of the present invention, the dilution step refers to the addition of a solution to the lysed biomass or lysate to reduce its dry matter concentration.

The diluted lysed biomass is then defined as a “solubilisate”.

Advantageously, the dilution step is carried out via the addition of an aqueous solution.

In one embodiment, the aqueous solution is water.

Preferably, dilution step c) is carried out on the lysate whose dry matter content is 5 to 30% by weight relative to the total weight of the lysate, preferably 12 to 25% by weight.

Also preferably, dilution step c) is carried out on the lysate whose C-phycocyanin concentration is 3% to 12% relative to the total weight of the dry matter.

The aqueous solution may additionally comprise one or more pH adjusting compounds. The term “pH adjusting compounds” means any organic or mineral compound that can modify the pH (acidity correcting agents, acids, bases, neutralizing agents or buffering agents). Examples of such compounds are sulfuric acid, acetic acid, citric acid, phosphoric acid, sodium citrate, potassium lactate, potassium malate, sodium chloride, disodium phosphate and potassium phosphate. The aqueous solution has an acidic or basic pH depending on the pH adjusting compound(s) present in the solution.

Preferably, step c) of dilution of the lysed biomass or lysate is carried out with an aqueous solution of pH less than or equal to 8, in particular between 0 and 6, preferably between 1 and 6, more preferably between 2 and 5. The pH of the aqueous solution to be added to the lysed biomass or lysate may be approximately 2, approximately 3, approximately 4 or approximately 5. Examples of acidic solutions that can be added to the lysed biomass are solutions comprising acids such as those described in the preceding paragraph.

Depending on the case, the solubilized solution has a pH close to neutrality with a pH between 6 and 8, a pH between 1 and 6 or a pH between 8 and 14.

Preferably, the solubilizer has a pH of less than 7, in particular between 1 and 6, more preferably between 2 and 5, even more preferably between 3 and 4.

Preferably, the solubilized material obtained has a dry matter content of 1 to 15% by weight relative to the total weight of the solubilized material, preferably 3 to 12%. by weight, more preferably 4 to 8% by weight.

Also preferably, the solubilized obtained according to the invention has a C-phycocyanin concentration of 0.1 to 12%, preferably 0.5 to 8%, more preferably 1 to 7%.

This dilution step c) can be carried out before or after the glycogen degradation step, i.e. before or after the resting step.

Preferably, dilution step c) is carried out after the resting step.

The method according to the invention thus comprises, according to this preferred embodiment, in order the successive steps of: a) harvesting the biomass by separation of the culture medium to obtain a crude ARU biomass, b) cell lysis of the crude biomass from step (a) to obtain a lysate, rest at acidic pH for at least 3 hours of the lysate, c) dilution of the lysate to obtain a solubilizer, and d) separation of the insolubles suspended in the solubilizer from step (c) to obtain a clarifier. d) Separation of the insolubles

In the context of the process according to the invention, the lysate or solubilized product undergoes a step d) of separation of the insoluble substances in suspension to obtain a clarified product.

This step d) of separation of insolubles is carried out after the glycogen degradation step, i.e. after the resting step.

The step of separating the insolubles d) from the lysate or solubilized can be carried out by any methods known to those skilled in the art. Particular mention will be made of frontal filtration methods and centrifugation.

According to the invention, step d) of separation of the insolubles is carried out on a lysate whose matter content is from 5 to 30% by weight relative to the total weight of the lysate, preferably from 10 to 25% by weight, more preferably 15 to 20% by weight; or more preferably on a solubilizer whose dry matter content is from 1 to 15% by weight relative to the total weight of the solubilizer, preferably from 3 to 12% by weight, more preferably from 4 to 8% by weight.

Preferably, according to the invention, step d) of separation of the insolubles is carried out on a lysate or a solubilizer with a pH of less than 7, preferably between 1 and 6, still preferably between 2 and 5, still still preferably between 3 and 4. The clarified material according to the invention preferably has a dry matter content of 0.1% to 5% by weight relative to the total weight of the clarified material, preferably of 0.5% to 4% by weight, more preferably of 1% to 3% by weight.

The clarified material according to the invention preferably has a C-phycocyanin concentration of 0.1 to 20 g/L. In the particular case where the process according to the invention does not include a step c), the clarified material according to the invention more preferably has a C-phycocyanin concentration of 7 to 19 g/L, even more preferably 12 to 18 g/L. In the opposite case, where the process according to the invention includes a step c), the clarified material according to the invention has a C-phycocyanin concentration of 0.1 to 12 g/L, preferably 0.5 to 8 g/L, more preferably 1 to 7 g/L.

Glycogen breakdown

The method according to the invention is characterized by a step of degradation of the glycogen using the endogenous enzymes of the microorganism, by keeping the raw biomass and/or the lysate and/or the solubilisate at rest (resting step) for at least 3 hours, during which the liquid medium comprising the glycogen is at an acidic pH.

In order for the degradation of glycogen by the endogenous enzymes of the microorganism to be effective during the resting stage, the endogenous enzymes must be active and must therefore not have been inactivated, for example, by a heating stage before said resting stage.

Time of rest stage

The resting for at least 3 hours is carried out at acidic pH, on the raw biomass, the lysate, the solubilized. In particular, according to the invention, the resting can be carried out before or after step b) of cell lysis, in other words on the raw biomass, the lysate and/or solubilized.

According to the invention, the resting step can advantageously be carried out with stirring of the raw biomass and/or the lysate and/or the solubilized material.

Preferably, the resting step is carried out only on the raw biomass which is preferably dried or thawed, the lysate or the solubilized. In the case of the lysate and/or the solubilized, the resting step is carried out if necessary after adjusting the pH to obtain the acidic pH sought for the resting step.

Preferably, according to the invention, the resting is carried out after step b) of cell lysis, in other words, preferentially on the lysate and/or solubilized and even preferentially on the lysate. Preferably, the resting step is carried out on a medium comprising lysed cells, i.e. on the lysate from step b) and/or, where appropriate, on the solubilized from step c).

Conditions of the rest stage

According to the invention, the resting step can be carried out on raw biomass, lysate and/or solubilized material immersed in darkness or exposed to natural or artificial light. Preferably, the resting step is carried out on raw biomass, lysate, solubilized material immersed in darkness.

According to the invention, the resting step is carried out on raw biomass, lysate, solubilized with or without aeration.

The “acid pH” of the medium comprising the glycogen during the resting stage is defined as a pH lower than 7, in particular between 1 and 6, preferably between 2 and 5, still preferably between 3 and 4.

For raw biomass, glycogen is in the intracellular medium of the cells constituting the biomass, which liquid medium is at an acidic pH.

Optionally, the method according to the invention further comprises a step of adjusting the pH of the raw biomass, the lysate and/or the solubilizer to the acidic pH of the resting step.

In this case, the pH of the raw biomass, the lysate and/or the solubilizer is adjusted to a pH lower than 7, in particular between 1 and 6, still preferably between 2 and 5, and always preferably between 3 and 4.

The reagents for adjusting the pH, i.e. acidifying or basifying the raw biomass, the lysate, the solubilisate can be added in a solid form or in the form of a solution. Advantageously, the pH of the raw biomass, the lysate, the solubilisate is adjusted by the addition of an acidic or basic solution, preferably in the form of an aqueous solution.

Examples of pH adjusting compounds are given above in the description of step c): sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, sodium hydroxide, sodium carbonate or sodium bicarbonate.

It is understood that a person skilled in the art will be able to determine whether a compound, or a solution, acidic or basic, must be added to the raw biomass, the lysate and/or the solubilizer in order to adjust the pH to the desired value.

When present, this pH adjustment step is carried out upstream of the resting step, on the raw biomass preferably thawed and/or dried, on the lysate, on the solubilized.

Advantageously, when present, this pH adjustment step is carried out on the lysate prior to the resting step and prior to dilution and separation steps c) and d).

The pH adjustment step may otherwise be concomitant with dilution step c) when it is present. In particular, if this step c) is present, the pH adjustment step may be carried out on the lysate concomitantly with this dilution step c) prior to the resting step. In this case, after dilution of the lysate, the solubilized product obtained has a pH of less than 7, in particular between 1 and 6, preferably between 2 and 5, and even more preferably between 3 and 4.

The resting stage can last up to a week or 7 days. Preferably, the resting stage lasts between 3 hours and a week, still preferably, from 3 hours to 48 hours, still preferably from 6 to 36 hours and always preferably from 10 to 24 hours.

Depending on the embodiment, the rest phase has a duration of several hours to several days, in particular approximately 3 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, between 48 and 72 hours, between 2 and 7 days, between 3 and 7 days, 4 days, 5 days or even 6 days.

According to one embodiment, the temperature of the raw biomass and/or the lysate and/or the solubilizer during the rest phase is maintained at a temperature between 15°C and 70°C, in particular between 15 and 50°C, preferably between 15 and 40°C, still preferably between 15 and 30°C.

Advantageously, the temperature of the raw biomass and/or the lysate and/or the solubilizer during this rest phase is maintained at a constant temperature, in particular at a temperature between 15°C and 70°C, in particular between 15°C and 50°C, preferably between 15 and 40°C, still preferably between 15 and 30°C.

According to another embodiment, the temperature of the raw biomass and/or the lysate and/or the solubilizer during the rest phase is maintained at a temperature below 15°C, preferably between 4°C and 15°C.

Advantageously, according to this other embodiment, the temperature of the raw biomass and/or the lysate and/or the solubilizer during this rest phase is maintained at a constant temperature, in particular at a temperature between 4°C and 15°C Note that for the resting stage, the closer we get to the optimum temperature and pH pair, the shorter the time required for the degradation of glycogen will be. For example, for a resting stage at a pH between 3 and 4 and a temperature between 15 and 30°C, the duration could be between 2 and 10 hours, whereas for a resting stage at a pH of around 6 and a temperature of around 10°C, the duration should be between at least 6 days and 7 days.

In the context of the present invention, the resting step is carried out with a liquid medium comprising the glycogen free of significant microbiological contamination. Such contamination can be avoided by adding preservatives to the liquid medium comprising the glycogen. These preservatives are well known to those skilled in the art and are in particular selected from sodium benzoate, potassium benzoate, calcium benzoate, benzoic acid, sodium diacetate, calcium propionate, sodium propionate, sodium nitrate, potassium sorbate, sodium sorbate, methyl gallate, propyl gallate, sodium ethylenediaminetetraacetate, methyl paraben, natamycin, propyl paraben and mixtures thereof.

Optional addition of exogenous enzymes

The method may also comprise a step of adding exogenous enzymes in addition to the resting step. This addition of exogenous enzymes makes it possible, as needed, to complete the degradation of the glycogen, in particular if the desired level of degradation is not reached. It is understood that this “supplement” means that the majority, i.e. at least 50%, of the degradation of the glycogen is the result of the implementation of the resting step independently of this possible addition.

“Enzymes” means proteins that enable the activation or acceleration of chemical or biological reactions. In the context of the present application, the term “exogenous enzymes” refers to enzymes that are not naturally produced by the cells or microorganisms of the treated unicellular red algae (URA) biomass. In particular, said exogenous enzymes are selected in particular from enzymes extracted from Aspergillus, Bacillus or Trichoderma.

The exogenous enzymes according to the invention have a glycogen degradation activity. These enzymes are well known to those skilled in the art and are in particular chosen from enzymes having a glucuronidase a1-4 activity, an activity glucosidase a1-4, glucosidase a1-6 activity, amylase activity. Note that it is possible to use one of these enzymes or a mixture of these enzymes. It has been found that these enzymes reduce the size of the glucosidic chains of the glycogen present in the medium which can then be eliminated later as well as its degradation by-products.

Among the enzymes having glucuronidase a1-4 and/or glucosidase a1-4 activity, mention will be made of pectinases known to degrade pectin, and in particular pectinases extracted from filamentous fungi such as Aspergillus, and more particularly pectinases extracted from Aspergillus aculeatus, such as the enzymes marketed under the name Pectinex® by the company Novozymes.

Examples of enzymes having a1-6 glucosidase activity are the pullulanases known to hydrolyze the a1-6 glucosidic bonds of pullulan, also known to remove starch branches. These are generally enzymes extracted from bacteria, in particular from the Bacillus genera. US patents 6,074,854 and US 5,817,498 and application WO2009/075682 describe such pullulanases extracted from Bacillus deramificans or Bacillus acidopullulyticus. Commercially available pullulanases are also known, in particular under the names Promozyme D2 and Novozym 26062 from Novozymes, or Optimax L1000 from DuPont-Genencor.

Among the enzymes with amylase activity known to degrade starch, many are known from the state of the art and are described in the literature and in particular in patent applications such as WO 2019/036721. Commercially available amylases are known, in particular under the names “Amylase AG XXL” (from Novozymes) or “Panzym® AG XXL” (from Eaton).

The enzymes can be used in their pure or enriched form, and optionally as a mixture with one or more excipients. The enzymes used in the process of the invention are in the form of powder or solution. In the latter case, the enzymes are preferably in solution in water.

The preferred conditions for implementing the exogenous enzymes are a pH of less than 7 and a reaction temperature of less than 60°C, preferably less than 50°C, and even less than 30°C. In particular, the temperature of the solution at which the exogenous enzymes are added and at which the enzymatic reaction occurs is between 4 and 60°C, preferably between 20 and 42°C and the pH of the solution is less than or equal to 5, preferably about 4.5. The exogenous enzymes can be added to the medium either in free form or immobilized on a support.

Exogenous enzymes can be added to the lysate in which case the enzymatic reaction by the exogenous enzymes takes place on the lysate and the resting step takes place previously on the raw biomass and/or previously on the lysate and/or concomitantly on the lysate.

According to a preferred embodiment, the exogenous enzymes are added after the resting step. In this case:

When the resting step is carried out on the raw biomass, exogenous enzymes can be added to the lysate and/or to the solubilizer and/or to the clarifier to complete the degradation of glycogen resulting from the resting of the raw biomass.

- when the resting step is carried out on the lysate, exogenous enzymes can be added to the lysate after the resting step, or to the solubilizer and/or to the clarifier to complete the degradation of glycogen resulting from the resting of the lysate;

- when the resting step is carried out on the solubilizer, exogenous enzymes can be added to the solubilizer after the resting step and/or to the clarifier, to complete the degradation of the glycogen resulting from the resting of the solubilizer;

According to an even more preferred embodiment, the resting step is carried out on the lysate and preferably, the possible addition of exogenous enzymes is done on the clarified or solubilized material.

This combination of rest and addition of exogenous enzymes makes it possible to reduce the quantities of exogenous enzymes used compared to the methods of the prior art.

Exogenous enzymes are thus added in a content of less than 0.01% by weight relative to the total weight of the raw biomass or lysate or solubilized or clarified to be treated, for an enzyme with an enzymatic activity equivalent to that of the Pectinex Ultra SP-L enzyme with an activity declared by the manufacturer of 3300 PGNU/g.

According to one embodiment, the content of added exogenous enzymes is less than or equal to 0.005%, by weight, preferably less than or equal to 0.0025%, more preferably less than or equal to 0.0001% by weight. Note that, if necessary, the concentration of exogenous enzymes is adjusted according to the activity of the exogenous enzyme selected to have an activity in the reaction medium. equivalent to that of a concentration according to the invention of Pectinex Ultra SP-L at 3300 PGNU/g.

The person skilled in the art will also know how to adapt the quantities of enzymes to be added during the process in order to increase the degradation of the glycogen present in the lysate, solubilized and/or clarified.

The glycogen degradation step with or without the addition of exogenous enzymes is considered sufficient, in particular to improve the subsequent filtration step(s), when at least 10%, preferably at least 50%, of the initial glycogen content in the raw biomass has been degraded, still preferably from 50% to 80% of the initial content. e) Concentration

The process according to the invention may further comprise a step e) of concentrating the clarified material using standard methods of removing water to obtain a concentrated aqueous extract.

The usual methods of removing water are known to those skilled in the art and include in particular filtration as well as evaporation at atmospheric pressure or under vacuum, atomization, infrared drying, refraction window drying, freeze-drying.

Preferably, step e) of the process according to the invention allows the concentration of the molecules of interest while preserving the essential constituents of the clarified material.

Depending on the concentration method chosen, concentration step e) makes it possible to eliminate all or part of the impurities such as solid residues, residual glycogen, oligomers and sugars resulting from the degradation of glycogen, present in the clarified material, in particular concentration by filtration.

Even more preferably, the biomass treatment process according to the invention comprises a step e) of concentration of the clarified material by filtration, in particular by tangential filtration such as ultrafiltration.

Preferably, the biomass treatment process according to the invention comprises a concentration step e) during which the clarified material is concentrated between 2 and 1000 times, more preferably between 20 and 60 times.

Preferably, concentration step e) is set to a pH lower than 7, preferably between 1 and 6, still preferably between 2 and 5, still still preferably between 3 and 4. In addition to the degradation supplement by addition of enzyme and/or concentration of the clarified material, the method according to the invention may comprise consecutive steps of purification of the clarified material and/or of the concentrated aqueous extract, in particular to purify the proteins in solution.

Product according to the invention

The product according to the invention comprises different organic materials depending on the treatment method applied, including water-soluble proteins including phycocyanins, sugars including glycogen degradation by-products (glucose oligomers) and possibly undigested residual glycogen and insolubles.

When present, phycocyanins may include acidic pH-resistant phycocyanins. Acidic pH-resistant phycocyanins are defined as phycocyanins that are stable at acidic pH, i.e. do not precipitate or lose their colour at acidic pH. Acidic pH resistance or stability may be measured as a loss of colour of less than 10% after a minimum of 10 minutes of exposure to acidic pH, i.e. pH below 7, particularly pH between 2 and 5. Acidic pH stability may also be measured by other methods such as monitoring the structure of the protein.

The presence of phycocyanins resistant to acidic pH comes from the implementation of steps d) and where appropriate e) at a pH lower than 7, in accordance with the extraction process described in WO2018/178334, preferably at a pH between 1 and 6, still preferably between 2 and 5 and even more preferably between 3 and 4.

According to a preferred embodiment, the product according to the invention comprises phycocyanins resistant to an acidic pH.

A concentrated aqueous extract is obtained by the process according to the invention comprising a step e) of concentration of the clarified material as described above. Conversely, a clarified material is obtained by the process according to the invention not comprising a step e) of concentration of the clarified material as described above.

Preferably, the product according to the invention does not comprise exogenous enzymes or comprises a concentration of exogenous enzymes undetectable by usual assay methods, in particular after precipitation of the proteins with acetonitrile, digestion with trypsin and then analysis by mass spectrometry (LC-MS-MS).

In particular, preferably, the product according to the invention does not comprise of exogenous enzymes selected from pectinases, amylases and pullulanases or comprises a concentration undetectable by standard assay methods, in particular by mass spectrometry.

If necessary, the product according to the invention is prepared so as to eliminate any impurities which would make it unfit for consumption, in particular human consumption.

The product according to the invention can also be formulated by means known to those skilled in the art to avoid the degradation of its constituents during its storage or subsequent use.

Finally, the product according to the invention, possibly formulated, can be packaged for its conservation and use, either in large volume containers, or in smaller containers, for example with volumes corresponding to a single use, called single dose, for human consumption. In this case, the container can be rigid, such as a glass ampoule, or flexible, such as a capsule suitable for consumption.

The sugar contents, in particular glucose, mannose and galactose (respectively called “hydrolyzed glucose”, “hydrolyzed galactose” and “hydrolyzed mannose”) of a product according to the invention are measured, after acid hydrolysis of the sample, by high-performance liquid chromatography via a Hi-Plex H+ column of the “ion exclusion/ligand exchange column” type and detection by refractometry (hereinafter “dosage of hydrolyzed sugars by HPLC-RID”). For this, 1.5 mL of the supernatant of a sample homogenized by vortexing are hydrolyzed with 1.5 mL of 2N sulfuric acid, at 110°C for 2 hours. The sample is then filtered (0.22 pm) and analyzed.

Note that the hydrolyzed glucose content thus measured includes both the glucose from undigested residual glycogen and the free glucose present in the product.

According to the invention, the free glucose content is determined by biochemical analysis, in particular using a YSI® biochemical analyzer following the manufacturer's recommendations.

Thus, to estimate the content of undigested residual glycogen in a product according to the invention, it is sufficient to subtract the free glucose content from the hydrolyzed glucose content.

The percentage of glycogen degradation is calculated as follows: (concentration of free glucose in the sample studied according to the invention / concentration maximum free glucose in an equivalent sample treated with 1% exogenous enzymes (volume of enzyme solution relative to total sample volume) x 100.

According to the invention, the protein content of a product according to the invention is determined by the DUMAS method. The sample is subjected to high-temperature combustion in a flow of pure oxygen, the nitrogen oxides produced are reduced by copper. After separation of the reaction by-products, the nitrogen is measured with a thermal conductivity detector, the result is expressed in quantity N (%). This percentage of nitrogen is then reduced to a quantity of proteins by applying the formula: N*6.25 (%) (ISO/TS 16634-2:2009).

Finally, to determine the C-phycocyanin content of a sample according to the invention, 500 μl of sample are mixed with a 100 mM Tris-CI buffer pH 7.5 (1.5 ml) and the absorbances at 652 and 620 nm measured via a Metier Toledo spectrophotometer. The C-phycocyanin concentration is then calculated by applying the following formula:

[Phycocyanin] in mg/mL = (0.162 x AQ20nm - 0.098 x AQ52nm) x Dilution

According to the invention, a degradation of glycogen of less than 10% by weight relative to the total glycogen is not considered to be sufficient degradation. In addition, a degradation of less than 10% does not improve the filtration of the extract. It is then considered that the degradation of glycogen does not work.

According to one embodiment, the clarified obtained according to the invention has an estimated undigested residual glycogen (g/L) / C-phycocyanin (g/L) ratio of less than 6, advantageously less than 4, preferably less than 3, more preferably less than 2.5, or even more preferably less than 1.

The clarified according to the invention has a concentration of total sugars measured by assay of hydrolyzed sugars by HPLC-RID of less than or equal to 40 g/L, in particular between 0.1 and 40 g/L, preferably between 1 and 20 g/L, more preferably between 3 and 11 g/L, and/or a concentration of hydrolyzed glucose of less than or equal to 20 g/L, preferably between 0.1 and 20 g/L, preferably between 1 and 10 g/L, still preferably between 2 and 5 g/L and/or a concentration of hydrolyzed galactose of less than or equal to 10 g/L, in particular between 0.01 and 10 g/L, preferably between 0.1 and 5 g/L, more preferably between 0.5 and 3 g/L, and/or a concentration of hydrolyzed mannose less than or equal to 10 g/L, in particular between 0.01 and 10 g/L, preferably between 0.1 and 5 g/L, more preferably between 0.5 and 3 g/L, and/or a concentration of free glucose less than or equal to 10 g/L, in particular between 0.01 and 10 g/L, preferably between 0.1 and 5 g/L, more preferably between 0.2 and 2 g/L.

The C-phycocyanin contents of the clarified advantageously range from 0.1 to 12 g/L, preferably 0.5 to 8 g/L, more preferably 1 to 7 g/L.

Preferably, the clarified material has a ratio (C-phycocyanins (g/L)/total sugars measured by assay of hydrolyzed sugars by HPLC-RID (g/L)) of at least 0.001, preferably from 0.005 to 120, more preferably 0.05 to 12, even more preferably 0.1 to 3.

According to another preferred embodiment of the invention, the clarified product comprises C-phycocyanin and total sugars in a ratio (C-phycocyanin (g/L)/total sugars measured by assay of hydrolyzed sugars by HPLC-RID (g/L)) of 0.005 to 120, in particular of 0.05 to 12, more preferably of 0.1 to 3.

In a particular embodiment where the concentrated aqueous extract has a high concentration of C-phycocyanin, the ratio (C-phycocyanin (g/L) / total sugars (g/L)) is at least 90, and may exceed 120.

Preferably, the clarified material has a ratio (C-phycocyanins (g/L) / hydrolyzed mannose (g/L)) of at least 0.01, preferably from 0.01 to 1200, more preferably 0.1 to 80, even more preferably 0.1 to 12.

Preferably, the clarified material has a ratio (C-phycocyanins (g/L) /hydrolyzed galactose (g/L)) of at least 0.01, preferably from 0.01 to 1200, more preferably 0.1 to 80, even more preferably 0.1 to 12.

The ratio (C-phycocyanin (g/L) / hydrolyzed glucose (g/L)) of the clarified is preferably less than 120 and preferably ranges from 0.005 to 120. Preferably, this ratio is between 0.05 and 12, more preferably 0.1 and 10.

Preferably, the concentrated aqueous extract according to the invention has a dry matter content of 2 to 1000 times higher than that of the clarified material from which it is derived, and even more preferably 20 to 60 times higher.

The C-phycocyanin contents of the concentrated aqueous extract advantageously range from 7 to 120 g/L, preferably 20 to 100 g/L, more preferably 40 to 80 g/L.

The concentrated aqueous extract according to the invention has a concentration of total sugars measured by assay of hydrolyzed sugars by HPLC-RID of less than or equal to 50 g/L, in particular between 1 and 50 g/L, preferably between 5 and 40 g/L, more preferably between 10 and 30 g/L, and/or a concentration of hydrolyzed glucose of less than or equal to 20 g/L, preferably between 0.1 and 20 g/L, preferably between 1 and 10 g/L, more preferably between 2 and 5 g/L and/or a concentration of hydrolyzed galactose of less than or equal to 15 g/L, in particular between 0.5 and 15 g/L, preferably between 1 and 13 g/L, more preferably between 3 and 8 g/L, and/or a concentration of hydrolyzed mannose of less than or equal to 15 g/L, in particular between 0.5 and 15 g/L, preferably between 1 and 13 g/L, more preferably between 3 and 8 g/L, and/or a free glucose concentration less than or equal to 15 g/L in particular between 0.01 and 15 g/L, preferably between 0.5 and 8 g/L, more preferably still between 1 and 4 g/L.

The weight ratio (C-phycocyanin (g/L) / hydrolyzed glucose (g/L)) of the concentrated aqueous extract ranges from 2 to 80. Preferably, this ratio is between 10 and 70, more preferably 15 and 60.

Preferably, the concentrated aqueous extract has a ratio (C-phycocyanin (g/L) / hydrolyzed mannose (g/L)) of at least 0.01, preferably from 0.01 to 1200, more preferably 0.1 to 80, even more preferably 0.3 to 12.

Preferably, the concentrated aqueous extract has a ratio (C-phycocyanin (g/L) /hydrolyzed galactose (g/L)) of at least 0.01, preferably from 0.01 to 1200, more preferably 0.1 to 80, even more preferably 0.3 to 12.

Preferably, the concentrated aqueous extract has a ratio (proteins (%) / estimated undigested residual glycogen (g/L)) of 0.2 to 6.0, more preferably 0.5 to 5.0, even more preferably 1.0 to 4.5.

Preferably, the concentrated aqueous extract has an estimated content of undigested residual glycogen (hydrolyzed glucose content (g/L) - free glucose content (g/L)) of 0.1 to 10 g/L, more preferably of 0.2 to 7 g/L, even more preferably 0.3 to 5 g/L.

Preferably, the concentrated aqueous extract has a ratio (proteins (%) /total sugars measured by determination of hydrolyzed sugars by HPLC-RID (g/L)) less than 1%, preferably between 1 and 0.01%, more preferably between 0.8 and 0.05%, even more preferably between 0.6 and 0.1%.

The present invention finally relates to a process for preparing a concentrated aqueous extract of an ARU biomass of phycocyanin-producing microalgae having a high glycogen content, said process comprising a treatment process with a glycogen degradation step, and a concentration step as defined above.

Use

The invention also relates to the use of all or part of a product obtained from a process according to the invention as a product for the preparation of pharmaceutical or food compositions, including foods or pharmaceutical, nutraceutical, food, cosmetic or industrial compositions. This includes in particular the use of a product obtained according to a process of the invention comprising phycocyanins for the preparation of pharmaceutical or food compositions.

According to one embodiment, a composition according to the invention comprises all or part of a product obtained with the method according to the invention and one or more excipients.

According to a preferred embodiment, the composition according to the invention is an acidic composition whose pH is less than or equal to 6, preferably less than or equal to 5, or even less than or equal to 4 and in particular between 2 and 4.

According to the invention, the term “acid composition” means any composition comprising all or part of a product obtained with a process according to the invention as well as a mineral or organic acid.

Mineral or organic acids that may be used in the compositions according to the invention are known to those skilled in the art. Among the mineral acids, mention will be made in particular of carbonic, phosphoric, hydrochloric, sulfuric, perchloric, sulfonic and nitric acids. Among the organic acids, mention will be made in particular of citric, lactic, malic, tartaric and succinic acids.

The acid compositions according to the invention may further comprise a vehicle which may comprise structural constituents associated with active compounds identified with regard to their nutritional contributions or for their properties beneficial to human or animal health.

Concerning compositions other than food compositions, they may be pharmaceutical, veterinary or cosmetic and further include one or more additives and/or active ingredients known and used in this type of indication.

The compositions according to the invention may be in any usual form known to those skilled in the art. In particular, solid, liquid, fluid, pasty or viscous forms and more particularly creams, gels, mousses, pastes will be mentioned. The compositions according to the invention may also be in the form of dry foods to be cooked, powders to be diluted, gelatinous compositions for food compositions.

In these solid compositions, all or part of the extract obtained according to the invention is preferably added in the form of powder. In which case, the product obtained from a process according to the invention is previously dried to take the form of a powder.

The liquid compositions are advantageously aqueous compositions in which all or part of the extract obtained according to the invention is dissolved.

According to a particular embodiment of the invention, the liquid composition may be a food composition and more specifically an acidic drink, whether carbonated or not. In particular, sodas, juices, sports drinks, exercise drinks, recovery drinks, etc. will be mentioned. The compositions of these drinks are well known to those skilled in the art and may comprise, in particular, sugars, mineral salts, food additives or dissolved gas. An acidic drink according to the invention may be an acidic drink of the prior art in which the coloring usually used has been replaced in whole or in part by a product comprising phycocyanins resistant to acid pH according to the invention.

The phycocyanin content in the compositions according to the invention will be in accordance with the concentrations usually applied in the intended field of application.

EXAMPLES

Example 1: Study of glycogen degradation with and without addition of exogenous enzymes at different pHs

The amount of free glucose over time in Galdieria sulphuraria biomass lysates at pH 3.75 without enzyme addition, at pH 6 without enzyme addition and at pH 6 with enzyme addition (1% of the volume of enzyme solution relative to the total volume of lysate, “Amylase AG XXL” from Novozymes), at 37 °C, was measured in time function (YSI® 2950).

These kinetics are presented in Figure 1.

Acidification of the lysate is achieved by adding a sufficient quantity of citric acid to increase the pH from 6 to 3.75.

The results show that at acidic pH 3.75, a greater amount of glucose is released than at pH 6. After 24 hours of incubation at pH 3.75 without the addition of enzyme, the free glucose in the sample even reaches a level identical to that in the sample in which an amount of exogenous enzyme was added.

It should be noted that glucose release also occurs at pH 6 in the absence of addition of exogenous enzymes; the kinetics are only slower (lower slope).

Example 2: Study of glycogen degradation without addition of exogenous enzymes at different pHs

The amount of free glucose over time in Galdieria sulphuraria biomass lysates (20% dry matter (DM) weight relative to the total lysate weight) at pH between 3 and 7 without enzyme addition and at room temperature (20°C) was measured over time (YSI® 2950).

These kinetics are presented in Figure 2.

Acidification and basification of the lysate are achieved respectively by adding a sufficient quantity of citric acid or sodium hydroxide.

The results show that in a given time, the higher the pH, the less glycogen is degraded. Performing glycogen digestion without adding exogenous enzymes on lysate shows activity mainly in the most acidic conditions. It is observed here that this activity is significant at pH 3 but slightly lower at pH 4. The activity for pH 5 to 7 is identical but slower than for lower pHs.

Example 3: Study of glycogen degradation at different temperatures with and without addition of exogenous enzymes

The amount of free glucose over time in Galdieria sulphuraria biomass lysates (20% dry matter (DM) weight relative to the total lysate weight) at pH 3.75 is studied for different temperatures (4 °C, 20 °C and 37 °C) without and with an enzyme (1% “Amylase AG XXL” from Novozymes) at pH 3.75 and at a temperature of 37°C (YSI® 2950).

The results are shown in Figure 3. They show glycogen digestion in free glucose even in the absence of enzyme addition. Notably, after 24 hours at 37°C, the amount of free glucose is the same with and without enzyme addition.

Example 4: Study of glycogen degradation at different temperatures without addition of exogenous enzymes

The amount of free glucose over time in Galdieria sulphuraria biomass lysates (20% by weight of dry matter (DM) relative to the total weight of the lysate) at pH 4 is studied for different temperatures (4 °C, 15 °C, 20 °C, 30 °C, 40 °C and 50 °C) without addition of exogenous enzymes (YSI® 2950).

The results are presented in Figure 4. They show that, in a given time, the higher the temperature, the greater the quantity of glycogen is digested during a resting stage according to the invention.

Example 5: Study of glycogen degradation at different stages of an extraction process

The amount of free glucose, at room temperature (20°C), in lysate (20% by weight of dry matter (DM) relative to the total weight of the lysate) and in the solubilized biomass of Galdieria sulphuraria at (7.5% DM relative to the total weight of the solubilized) at pH 3.75, without the addition of exogenous enzymes is studied (YSI® 2950). In this context, the kinetics of glucose release at room temperature (20°C), in lysate of biomass of Galdieria sulphuraria at pH 3.75 with the addition of exogenous enzymes (1% of the volume of enzyme solution relative to the total volume of lysate “Amylase AG XXL”, Novozymes) is taken as a control.

These kinetics are presented in Figure 5.

From the results obtained and by extrapolation, it is possible to conclude that, even without the addition of enzyme, in the case of the lysate, after 24 hours, all the digestible glycogen will be degraded and released in the form of free glucose. On the other hand, for the solubilized, the reaction seems slower and is still not complete after 24 hours.

It should be noted that the samples studied all show a glycogen degradation of more than 10% and thus good filterability after glycogen digestion whether they were prepared with or without the addition of exogenous enzymes.

Example 6: Study of glycogen degradation in the prior art

In patent WO2020/144330, example 2 shows the degradation of the glycogen in a crude solution of phycocyanins in the presence and absence of exogenous enzymes.

For this, a biomass of Galdieria sulphuraria is produced then recovered/harvested. The cells are then lysed then diluted in order to obtain an aqueous extract, a solubilizer. A clarifier is then obtained from this aqueous extract by separating the solids by filtration on a 0.22pm filter.

The amount of free glucose, at room temperature (20°C), in Galdieria sulphuraria biomass clarified (7.5% by weight of DM relative to the total weight of the clarified) at pH 4, without and with a step of addition of exogenous enzymes (0.1% of the volume of enzymatic solution relative to the total volume of clarified “Amylase AG XXL” from Novozymes, “Pectinex Ultra SP-L” from Novozymes or “BAN 480 L” from Novozymes) is studied (YSI 2700 Biochemestry Analyzer).

The results are presented in Figure 6.

From the results obtained, it is possible to conclude that without the addition of exogenous enzymes, with the clarified material left to rest for 95 hours, the glycogen is still not digested enough (less than 10% degradation). On the other hand, with the addition of an enzyme, after 95 hours, all of the glycogen is digested.

Example 7: Study of glycogen degradation at all stages of an extraction process

The amount of free glucose, at room temperature (20°C), in lysate (20% by weight of DM relative to the total weight of the lysate), solubilized and clarified of Galdieria sulphuraria biomass at (7.5% by weight of DM relative to the total weight of the solubilized or clarified) at pH 4 without the addition of exogenous enzymes is studied (YSI® 2950). The clarified is obtained after filtration of the solubilized on a 0.22pm filter.

In this context, the kinetics of glucose release at room temperature (20°C), in lysate, solubilized and clarified biomass of Galdieria sulphuraria at pH 4 with addition of exogenous enzymes (1% of the volume of enzymatic solution relative to the total volume of lysate “Amylase AG XXL”, Novozymes) are used to know the maximum concentration of free glucose in the sample.

These data made it possible to calculate the percentages of degradation (digestion) of glycogen which are presented in Figure 7.

From the results obtained, we see that, without the addition of exogenous enzymes, after 48 hours, more than 10% of the glycogen is digested when the lysate and the solubilized are kept at rest but not the clarified. Example 8: Characterization of an extract prepared according to the invention

A Galdieria sulphuraria biomass lysate (20% by weight of DM relative to the total weight of lysate) is maintained at acidic pH of 3.7 for 12 hours, at 25 °C.

An extract is prepared from this lysate. To do this, the lysate is washed with a quantity of water representing in total less than 4 times the total volume of lysed biomass. This volume of water is split into 3 fractions to carry out 3 successive washes of the lysed biomass. The wash water is recovered, in accordance with the teaching of patent application WO2020/161280. The product obtained is then filtered on a hollow fiber membrane having a porosity of 70 kDa (clarifiate) with a final diafiltration step and the filtrate is recovered, in accordance with the teaching of patent application WO2020/144330 (concentrated aqueous extract).

The concentrated aqueous extract and the clarified thus obtained are analyzed to know the content of total sugars, hydrolyzed mannose, hydrolyzed galactose and hydrolyzed glucose by HPLC-RID, the content of free glucose at YSI® 2950, the content of phycocyanin as well as the content of proteins.

The results obtained are presented in Table 1 below.

Table 1

Claims

1. A method for treating a biomass of unicellular red algae (URA) of the genus Galdieria, said method for treating URA biomass comprising the steps of: a) harvesting the biomass by separating the culture medium to obtain a crude URA biomass, b) cell lysis of the crude biomass from step (a) to obtain a lysate, c) optionally diluting the lysate from step (b) to obtain a solubilizer, and d) separating the insolubles suspended in the lysate from step (b) or the solubilizer from step (c) to obtain a clarifier, characterized in that it comprises a step of degrading the glycogen by keeping the crude biomass and/or the lysate and/or, where appropriate, the solubilizer at rest for at least 3 hours (resting step), during which the liquid medium comprising the glycogen is at an acidic pH.

2. Method according to claim 1, characterized in that the resting step lasts between 3 hours and one week.

3. Method according to claim 1 or 2, characterized in that the resting step is carried out at a temperature between 15°C and 70°C.

4. Method according to one of claims 1 to 3, characterized in that the resting step is carried out on the lysate and/or the solubilized product where appropriate, preferably on the lysate.

5. Method according to one of claims 1 to 4, characterized in that it comprises, prior to the resting step, a step of adjusting the pH of the raw biomass, the lysate and/or where appropriate the solubilizer to a pH lower than 7, in particular between 1 and 6, preferably between 2 and 5, more preferably between 3 and 4.

6. Method according to one of claims 1 to 5, characterized in that the method further comprises a step e) concentration of the clarified material to obtain an extract concentrated aqueous.

7. Product capable of being obtained by the process according to one of claims 1 to 6.

8. Product according to claim 7, characterized in that it comprises an estimated content of undigested residual glycogen of 0.1 to 10 g/L, more preferably of 0.2 to 7 g/L, even more preferably 0.3 to 5 g/L.

9. Product according to claim 7 or 8, characterized in that it comprises a concentration of hydrolyzed mannose less than or equal to 15 g/L.

10. Product according to one of claims 7 to 9, characterized in that it comprises a concentration of hydrolyzed galactose less than or equal to 15 g/L.

1 1. Product according to one of claims 7 to 10, characterized in that it comprises a protein (%) / estimated undigested residual glycogen ratio measured by assay of hydrolyzed sugars by HPLC-RID (g/L) of 0.2 to 6.0, more preferably 0.5 to 5.0, even more preferably 1.0 to 4.5.

12. Product according to one of claims 7 to 11, characterized in that it comprises a protein (%) / total sugars ratio measured by assay of hydrolyzed sugars by HPLC-RID (g/L) of less than 1%, preferably between 1 and 0.01%, more preferably between 0.8 and 0.05%, even more preferably between 0.6 and 0.1%.

13. Product according to one of claims 7 to 12, characterized in that it comprises a C-phycocyanin content of between 0.1 and 20 g/L.

14. Nutraceutical, food or cosmetic composition comprising a product according to one of claims 7 to 13.

15. Composition according to claim 14 characterized in that it comprises a mineral or organic acid.