Method for degrading glycogens present in biomass of genus Galdiella

Abstract

The present invention relates to a treatment method for degrading glycogens present in biomass of unicellular red algae (URA), in particular of the genus Galdiella, and to the products thus obtained.

Description

Technical Field

[0001] The present invention relates to a method for treating glycogen present in biomass of a single-celled red algae (URA) of the genus Galdieria, and to the aqueous products obtained therefrom. Background Technology

[0002] Unicellular red algae (URA), or Rhodophyte, are characterized by the presence of pigments within their cells. In addition to chlorophyll and carotenoids, unicellular red algae also produce phycobiliproteins. These natural pigments, derived from photosynthesis, are classified into four types: allophycocyanin, C-phycocyanin, phycoerythrin, and phycoerythrocyanin.

[0003] Phycocyanin has properties that are beneficial to human and animal health and is now used in multiple fields, including the pharmaceutical, cosmetic and food industries.

[0004] Microalgae belonging to the class Cyanidiophyceae (and more specifically to the genera *Cyanidioschyzon*, *Cyanidium*, and *Gardilia*) are of particular interest for the production of phycocyanin. The production of biomass from single-celled red algae is well known to those skilled in the art, specifically for the production of molecules of interest, particularly proteins such as phycocyanin. Methods for the production and extraction of said phycocyanin are described in the literature (WO2017 / 093345, WO2019 / 228947, WO2018 / 178334, WO2020 / 161280).

[0005] However, it is also known that this type of microalgae (particularly *Galdicella*) uses glycogen as its primary storage sugar. This glycogen is a polymer of α(1→4) glucose branched by α-(1→6) bonds. Glycogen from *Galdicella* microalgae has the specific characteristic of possessing a high proportion of these branches, with approximately 7% to 18% of the branched glucose molecules randomly distributed along the molecule. This molecular structure gives the glycogen a spherical shape, making it soluble in water (Martinez-Garcia et al., *Int J Biol Macromol*. *International Journal of Biological Macromolecules* (2016) 89:12-8). Furthermore, if *Galdicella* microalgae possess enzymes for synthesizing highly branched glycogen, they also possess enzymes for degrading it (Martinez-Garcia et al., *Int J Biol Macromol*. *International Journal of Biological Macromolecules* (2016) 89:12-18). These enzymes are intracellular enzymes. The intracellular medium of these microalgae has a pH between 6.3 and 7.1 (Miyagishima et al., Plant Cell Physiol. 62(6): 926-941 (2021)). Therefore, these enzymes are known to be active within this precise pH range.

[0006] To prepare aqueous extracts of microalgae, such as phycocyanin extracts from *Galdieriasulphuraria*, a filtration step may be necessary. Because glycogen is readily soluble in cold water, it has been found to coexist with hydrophilic compounds of interest, such as phycocyanin, in the aqueous fraction. Filters used to purify phycocyanin retain all or part of the glycogen, thereby increasing the viscosity of the residue. This imposes technical limitations on the method, such as pressure buildup, reduced flow rate, and clogging, especially when using tangential filtration membranes. On the other hand, if glycogen is not eliminated, the resulting extract will have high glycogen content, high viscosity, and low phycocyanin content.

[0007] Glycogen is known to be resistant to certain enzymes. However, methods have been developed that involve adding suitable exogenous enzymes to degrade it (WO2020 / 144330).

[0008] If an enzyme cannot be completely eliminated and / or inactivated, the enzyme added for the preparation of the dietary product must be listed in the product's composition. Such non-eliminable substances correspond to additives or processing aids that may cause marketing and / or formulation difficulties.

[0009] Other methods for preparing aqueous extracts from biomass, specifically from single-celled red algae of the genus *Gardinidia*, have been described in the literature (Moon et al., *Korean J. Chem.*, 2016, 31, 3, pp. 490-495). However, these methods are difficult to apply on an industrial scale because they require the addition of large amounts of ammonium sulfate, resulting in effluents rich in undesirable contaminants for both ecological and economic reasons, or require the use of very expensive instruments such as chromatographs, or involve a series of steps that are difficult to implement on a larger scale than in the laboratory. Furthermore, none of these methods can selectively remove glycogen from biomass or aqueous microalgae extracts.

[0010] Therefore, there is a need for a method for degrading glycogen present in the biomass of single-celled red algae (specifically, in the biomass of microalgae of the genus *Gardilia*) whose main reserve sugar is glycogen, while overcoming the problems of the prior art, and specifically the problems stated above. Summary of the Invention

[0011] According to a first aspect of the present invention, the inventors have developed a method for processing biomass of single-celled red algae (URA) of the genus *Gardinidia*, the URA biomass processing method comprising the following steps:

[0012] a) Harvesting biomass through a separation medium to obtain the original biomass for URA.

[0013] b) The crude biomass from step (a) is subjected to cell lysis to obtain lysate.

[0014] c) Optionally, the lysate from step (b) can be diluted to obtain a solubilizer, and

[0015] d) Separate the insoluble fractions suspended in the pyrolyte from step (b) or the solubilized fraction from step (c) to obtain a clarified fraction.

[0016] The method includes a glycogen degradation step in which the original biomass and / or lysate and / or solubilized material is left to stand for at least 3 hours, during which time the liquid medium containing glycogen is at an acidic pH.

[0017] Surprisingly, glycogen degradation by endogenous enzymes of microorganisms is efficient over a wide pH range, not limited to the intracellular pH of the microorganisms, but optimal for more acidic pH levels (particularly between 2 and 5). Still surprisingly, intracellular enzymatic degradation works in treated biomass (such as raw biomass, thawed raw biomass, or dried raw biomass), and also in lysates and solubilizers. The invention also relates to products obtainable by the method according to the invention.

[0018] Advantageously, the method according to the invention makes it possible to reduce or even eliminate the addition of exogenous enzymes suitable for degrading ARU glycogen, thereby reducing the addition of substances that must be classified as additives and / or processing aids.

[0019] The method according to the invention can also be applied under conditions ranging from the mildest to the most stringent in terms of time, temperature, and pH, and is therefore adaptable to the stability of the molecules of interest to be extracted. Attached Figure Description

[0020] Figure 1 The free glucose concentration over time in *Galdirius sulfadiazine* biomass lysate is shown at 37°C and at pH 3.75 and pH 6, without the addition of exogenous enzymes (SE: no enzyme) and with 1% exogenous enzymes in the lysate (E: with enzyme).

[0021] Figure 2 The diagram shows the change in free glucose concentration over time in the biomass lysate of *Galdirius sulfadiazine* at room temperature ("amb", i.e., 20°C) in the absence of exogenous enzymes and at different pH levels.

[0022] Figure 3 The figure shows the change in free glucose concentration over time in the biomass lysate of *Galdirius thiophilus* at different temperatures and pH 3.75, with or without exogenous enzymes (WE: no enzyme; E: with enzyme).

[0023] Figure 4 The figure shows the change in free glucose concentration over time in the biomass lysate of *Galdirius thiophilus* at different temperatures and pH 3.75 in the absence of exogenous enzymes.

[0024] Figure 5 The changes in free glucose concentration over time are shown in the biolysate and solubilized product of *Galdirius thiophilus* at pH 3.75 and 20°C in the absence of exogenous enzymes, and in the biolysate of *Galdirius thiophilus* at pH 3.75 in the presence of exogenous enzymes (WE: no enzyme; E: with enzyme).

[0025] Figure 6 The percentage of glycogen digestion in clarified *Galdirius sulfadiazine* biomass changes over time at pH 4 and 20°C, in the absence or presence of exogenous enzymes.

[0026] Figure 7 The percentage of glycogen digestion in the biomass lysate, solubilized fraction, and clarified fraction of *Galdirius sulfadiazine* at pH 4 and 20°C in the absence of exogenous enzymes is shown as a function of time (WE: no enzymes). Detailed Implementation

[0027] Definitions

[0028] In the context of this invention, the term "biomass" refers to a collection of microalgal cells preferably produced by fermentation in a bioreactor. The biomass can be considered as a mass of single-celled organisms.

[0029] Biomass can undergo various treatments and can be virgin biomass, pyrolyzed biomass, thawed virgin biomass, and / or dried virgin biomass.

[0030] In the context of this application, it should be understood that the properties of biomass correspond to the average of the properties of all the cells constituting the biomass. In other words, lysed biomass is biomass containing at least 50% lysed cells relative to the total number of cells, and the original biomass may contain cells lysed due to the harvesting step. As long as the number of lysed cells remains a minority (i.e. less than 50%) relative to the number of unlysed cells, it is not considered lysed biomass.

[0031] The term "raw biomass" refers to biomass obtained after harvest, that is, biomass that may be thawed and / or dried after the fermentation broth has been recovered and the cells separated from at least a portion of the culture medium.

[0032] The term "lysed biomass" or "lysate" refers to microalgal biomass in which at least 50% of the cells are lysed, preferably at least 70%, more preferably at least 80%, 85%, 90%, 95%, up to 100% of the cells are lysed.

[0033] The term "thawed raw biomass" refers to raw biomass that may have been frozen for storage and / or transportation reasons and then thawed to a temperature suitable for processing according to the present invention, specifically a temperature suitable for the glycogen degradation step of the biomass according to the present invention.

[0034] According to the present invention, the term "dried raw biomass" refers to raw microalgal biomass that has been dried using methods known to those skilled in the art, and whose moisture content relative to the total weight of the biomass is less than 10%, preferably less than 7%, and more preferably between 5% and 1%. Known drying methods include natural air drying, spray drying, fluidized bed drying, drum dryer drying, and freeze drying.

[0035] The term "solubilizer" refers to pyrolyzed biomass or pyrolytes that have undergone a dilution process with an aqueous solution of neutral, acidic, or alkaline pH.

[0036] The term "clarification fraction" refers to the aqueous extract obtained after separating insoluble matter suspended in lysates or solubilized fractions.

[0037] The term "setting" refers to a stage during which the chemical properties of the raw biomass, pyrolytes, and / or solubilizers, preferably dried or frozen, are not altered by the addition or extraction of one or more components. Setting does not preclude the mixing (i.e., stirring) of the raw biomass, pyrolytes, solubilizers, and / or clarifying fractions under non-destructive conditions that do not affect the chemical properties of the raw biomass, pyrolytes, and / or solubilizers.

[0038] The term "liquid medium containing glycogen" describes the intracellular medium of cells that constitutes the liquid phase (if applicable) of the original biomass and / or lysates, solubilizers and / or clarified fractions.

[0039] The term "biomass processing" refers to any method applied to biomass, specifically methods used to alter its physical / chemical properties, extract molecules of interest, and / or purify them.

[0040] It should be noted that the given numerical range is intended to include all intermediate numbers (for example, the range from 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32 and 5).

[0041] Please note that all numerical values ​​given refer to actual values ​​and approximate values ​​estimated based on general conventions; that is, the last digit indicated corresponds to the precision of the measurement. In the absence of precise error limits, rounding conventions should be used to estimate the maximum error of the specified last digit.

[0042] Process according to the invention

[0043] The method according to the present invention is a method for processing biomass of single-celled red algae of the genus *Gardinidia* (URA), the URA biomass processing method comprising the following steps:

[0044] a) Harvesting biomass through a separation medium to obtain the original biomass for URA.

[0045] b) The crude biomass from step (a) is subjected to cell lysis to obtain lysate.

[0046] c) Optionally, the lysate from step (b) can be diluted to obtain a solubilizer, and

[0047] d) Separate the insoluble fractions suspended in the pyrolyte from step (b) or the solubilized fraction from step (c) to obtain a clarified fraction.

[0048] The method includes a glycogen degradation step in which the original biomass and / or lysate and / or solubilized material is left to stand for at least 3 hours, during which time the liquid medium containing glycogen is at an acidic pH.

[0049] Ideally, temperature, time, and pH conditions should be specifically adjusted throughout the method to prevent the degradation of target compounds, such as phycocyanin, in the aqueous extract, while promoting glycogen degradation during the settling phase.

[0050] In one embodiment, a glycogen degradation step is performed on the virgin biomass. This embodiment then sequentially includes the following steps:

[0051] a) Harvesting biomass through a separation medium to obtain the original biomass for URA.

[0052] The raw biomass was allowed to stand for at least 3 hours, during which time the liquid medium containing glycogen was at an acidic pH.

[0053] b) The crude biomass from step (a) is subjected to cell lysis to obtain lysate.

[0054] c) Optionally, dilute the lysate from step (b) to obtain a solubilizer.

[0055] d) Separate the insoluble fractions suspended in the pyrolyte from step (b) or the solubilized fraction from step (c) to obtain a clarified fraction, and

[0056] Optionally, the step of adjusting the pH of the original biomass.

[0057] In this embodiment, glycogen is present in the intracellular medium of the cells constituting the biomass, the liquid medium being at an acidic pH, and the original biomass is preferably dried or thawed. In the following two embodiments, after cell lysis, glycogen is present in the liquid phase of the lysate and, where applicable, in the solubilizer.

[0058] In one embodiment, a glycogen degradation step is performed on the lysate. This embodiment then sequentially includes the following steps:

[0059] a) Harvesting biomass through a separation medium to obtain the original biomass for URA.

[0060] b) The crude biomass from step (a) is subjected to cell lysis to obtain lysate, which is then allowed to stand in a liquid medium containing glycogen at an acidic pH for at least 3 hours.

[0061] c) Optionally, dilute the lysate from step (b) to obtain a solubilizer.

[0062] d) Separate the insoluble fractions suspended in the pyrolyte from step (b) or the solubilized fraction from step (c) to obtain a clarified fraction, and

[0063] Optionally, a step of adjusting the pH of the original biomass or lysate.

[0064] It may be necessary to adjust the pH of the liquid medium of the raw biomass and / or pyrolyte to obtain the acidic pH required for the pyrolyte settling step.

[0065] In another embodiment, a glycogen degradation step is performed on the solubilizer. This embodiment then includes the following steps in sequence:

[0066] a) Harvesting biomass through a separation medium to obtain the original biomass for URA.

[0067] b) Perform cell lysis on the crude biomass from step (a) to obtain lysate; c) Dilute the lysate from step (b) to obtain solubilizer.

[0068] Allow the solubilized material to stand for at least 3 hours, during which time the liquid medium containing glycogen is at an acidic pH.

[0069] d) Separate the insoluble matter suspended in the solubilized fraction from step (c) to obtain a clarified fraction, and

[0070] Optionally, the step of adjusting the pH of the original biomass, pyrolysate, or solubilized material.

[0071] It may be necessary to adjust the pH of the liquid medium containing the raw biomass, pyrolysate, and / or solubilizer to obtain the acidic pH required for the solubilizer settling step.

[0072] a) Harvesting of the biomass

[0073] The biomass according to the present invention is biomass of single-celled red algae (URA), and more specifically, biomass of microalgae that produce phycocyanin and have high glycogen content.

[0074] These microalgae belong to the class Rhodophyta, which includes the genera *Galdicella*, *Chlorophyta*, and *Chlorophyta fissicans*. Preferably, the microalgae belong to the genus *Galdicella*.

[0075] Microalgae of the genus *Galdieria* include *Galdieria daedala*, *Galdieria maxima*, *Galdieria partita*, *Galdieria thorophilic*, *Galdieria phlegrea*, *Galdieria javensis*, *Galdieria yellowstonensis*, and *Galdieria sp*. Preferably, the biomass according to the present invention is biomass of *Galdieria thorophilic*.

[0076] Methods for producing biomass from single-celled red algae (URA) (specifically from the genus *Gardilia*) are well known to those skilled in the art. In the context of this invention, microalgae can be cultured in containers suitable for microbial growth (also known as bioreactors, bioreactors, or fermenters) using any known culture techniques.

[0077] According to the present invention, the biomass of single-celled red algae is obtained from microalgae cultured industrially in a high-capacity reactor, preferably to obtain a fermentation broth containing a high density of microorganisms. For the purposes of this application, "high density" means a quantity corresponding to more than 50 g dry matter / L fermentation broth, preferably more than 100 g / L. Examples of single-celled red algae cultures are described in patent applications WO2017 / 050917, WO2017 / 050918, WO2017 / 093345 and WO2019 / 228947. It should be understood that those skilled in the art will be able to determine the optimal parameters and conditions for culturing microorganisms, such as temperature, light, culture time, or the nature and amount of nutrients to be supplied. The method according to the present invention includes step a) of harvesting the biomass.

[0078] Once the microorganisms have been cultured and biomass has been obtained, they are harvested to produce raw biomass. Single-celled red algae can be harvested using any technique known to those skilled in the art, including filtration (possibly by gravity or under reduced pressure), decanting, gravity filtration after sedimentation, or centrifugation.

[0079] The method according to the invention includes step a) of harvesting biomass corresponding to the recovery of fermentation broth, followed by separating the biomass cells from at least a portion of the culture medium.

[0080] This step produces raw biomass. Raw biomass harvested in this way can also preferably be washed with water to remove certain soluble impurities.

[0081] The raw biomass obtained after harvesting and optionally after one or more washings contains at least 70% and at most 90% water, preferably 75% to 88% water.

[0082] Preferably, the original biomass according to the invention has a dry matter content of 5% to 30% by weight relative to the total weight of the original biomass, typically and more preferably 10% to 25% by weight, and more preferably 10% to 20% by weight.

[0083] Preferably, the crude biomass according to the invention has a C-phycocyanin concentration between 3% and 12%.

[0084] b) Cell lysis

[0085] The method for processing single-celled red algae biomass according to the present invention includes step b) of lysing the original biomass from step a) to obtain lysate.

[0086] Preferably, cell lysis step b) is performed on the original biomass having a dry matter content of 5% to 30% by weight, preferably 10% to 25% by weight, and more preferably 10% to 20% by weight relative to the total weight of the original biomass.

[0087] More preferably, the crude biomass according to the invention, having a C-phycocyanin concentration between 3% and 12%, undergoes a cell lysis step (b).

[0088] Cell lysis can be performed by any lysis method known to the technician, specifically by enzymatic, mechanical, and / or chemical means.

[0089] In the context of this invention, prior to the cell lysis step b), the original biomass may have undergone washing, freezing, thawing, drying, and / or rehydration. In other words, in the context of this invention, the original biomass may be thawed and / or dried biomass.

[0090] In a preferred embodiment of mechanical pyrolysis, the mechanical apparatus that can be used according to the invention includes a ball mill, a high-shear mixer, a high-pressure homogenizer, a pin mill, an impact mill, or an ultrasonic or pulsed electric field. As equipment for carrying out these methods, we refer to the following apparatus: ball mill: Netzsch Discus-100 or WAB ECM-AP60; high-pressure homogenizer: GEA Ariete; high-shear mixer: Silverson 700-X; pin mill: Hosokawa Contraplex; and impact mill: Netzsch Condux.

[0091] Preferably, cell lysis is performed by mechanical lysis, more preferably by grinding, and even more preferably by ball milling.

[0092] Preferably, the obtained pyrolyte has a dry matter content of 5% to 30% by weight, more preferably 10% to 25% by weight, and more preferably 10% to 20% by weight relative to the total weight of the pyrolyte.

[0093] Preferably, the obtained lysate has a C-phycocyanin concentration of 3% to 12% relative to the total dry matter weight.

[0094] The pyrolysis step b) can be performed before or after the glycogen degradation step, i.e. before or after the settling step, preferably before.

[0095] c) Optional dilution of the lysate

[0096] According to the present invention, the pyrolyzed biomass or pyrolytes may optionally undergo a dilution step.

[0097] In the context of this invention, the dilution step refers to adding a solution to pyrolyzed biomass or pyrolysate to reduce its dry matter concentration.

[0098] Diluted pyrolyzed biomass is thus defined as a "solubilizer".

[0099] Advantageously, the dilution step is performed by adding an aqueous solution.

[0100] In one implementation, the aqueous solution is water.

[0101] Preferably, step c) involves diluting the pyrolyte having a dry matter content of 5% to 30% by weight, preferably 12% to 25% by weight, relative to the total weight of the pyrolyte.

[0102] Preferably, a dilution step c) is performed on the lysate having a C-phycocyanin concentration of 3% to 12% relative to the total weight of the dry matter.

[0103] Aqueous solutions may also contain one or more pH-adjusting compounds. The term "pH-adjusting compound" refers to any organic or inorganic compound (acidity corrector, acid, base, neutralizer, or buffer) used to change the pH. 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. An aqueous solution may have an acidic or alkaline pH, depending on the pH-adjusting compound present in the solution.

[0104] Preferably, step c) involves diluting the pyrolyzed biomass or pyrolyte with an aqueous solution with a pH less than or equal to 8, specifically between 0 and 6, preferably between 1 and 6, and more preferably between 2 and 5. The pH of the aqueous solution to be added to the pyrolyzed biomass or pyrolyte may be about 2, about 3, about 4, or about 5. Examples of acidic solutions that can be added to the pyrolyzed biomass are solutions containing acids (such as those described in the preceding paragraphs).

[0105] Depending on the situation, the solubilizer has a near-neutral pH, a pH between 6 and 8, a pH between 1 and 6, or a pH between 8 and 14.

[0106] Preferably, the solubilizer has a pH below 7, specifically between 1 and 6, more preferably between 2 and 5, and even more preferably between 3 and 4.

[0107] Preferably, the obtained solubilizer has a dry matter content of 1% to 15% by weight, more preferably 3% to 12% by weight, and more preferably 4% to 8% by weight relative to the total weight of the solubilizer.

[0108] More preferably, the solubilizer obtained according to the present invention has a C-phycocyanin concentration of 0.1% to 12%, preferably 0.5% to 8%, and more preferably 1% to 7%.

[0109] The dilution step c) can be performed before or after the glycogen degradation step, i.e. before or after the settling step.

[0110] Preferably, the dilution step c) is performed after the settling step.

[0111] Therefore, according to this preferred embodiment, the method according to the invention sequentially includes the following steps:

[0112] a) Harvesting biomass through a separation medium to obtain the original biomass for URA.

[0113] b) Perform cell lysis on the crude biomass from step (a) to obtain lysate, and allow the lysate to stand at an acidic pH for at least 3 hours.

[0114] c) Dilute the lysis product to obtain a solubilizer, and

[0115] d) Separate the insoluble matter suspended in the solubilized material from step (c) to obtain a clear fraction.

[0116] d) Separation of insoluble matter

[0117] As part of the method according to the invention, the pyrolyte or solubilizer undergoes step d) to separate the suspended insoluble matter, thereby obtaining a clarified fraction.

[0118] The insoluble matter separation step (d) is carried out after the glycogen degradation step, that is, after the settling step.

[0119] The step of separating insoluble matter (d) from pyrolytes or solubilized products can be performed by any method known to those skilled in the art. These methods include pre-filtration and centrifugation.

[0120] According to the present invention, an insoluble matter separation step d) is performed on a pyrolyte having a dry matter content of 5% to 30% by weight, preferably 10% to 25% by weight, more preferably 15% to 20% by weight relative to the total weight of the pyrolyte; or more preferably, this step is performed on a solubilizer having a dry matter content of 1% to 15% by weight, preferably 3% to 12% by weight, more preferably 4% to 8% by weight relative to the total weight of the solubilizer.

[0121] Preferably, according to the invention, a step d) for separating insoluble matter is performed on lysates or solubilizers having a pH of less than 7, preferably between 1 and 6, even more preferably between 2 and 5, and still more preferably between 3 and 4.

[0122] The clarified fraction according to the invention preferably has a dry matter content of 0.1% to 5% by weight, more preferably 0.5% to 4% by weight, and more preferably 1% to 3% by weight relative to the total weight of the clarified fraction.

[0123] The clarified fraction according to the invention preferably has a C-phycocyanin concentration of 0.1 g / L to 20 g / L. In certain cases where the method according to the invention does not include step c), the clarified fraction according to the invention has a C-phycocyanin concentration of 7 g / L to 19 g / L, or even more preferably 12 g / L to 18 g / L. In the opposite case where the method according to the invention includes step c), the clarified fraction according to the invention has a C-phycocyanin concentration of 0.1 g / L to 12 g / L, preferably 0.5 g / L to 8 g / L, and more preferably 1 g / L to 7 g / L.

[0124] Glycogen degradation

[0125] The method according to the invention is characterized by a step of using endogenous enzymes of microorganisms to degrade glycogen, wherein the original biomass and / or lysate and / or solubilized material is kept in a static state (static step) for at least 3 hours, during which time the liquid medium containing glycogen is in an acidic pH.

[0126] In order for the endogenous enzymes of microorganisms to effectively degrade glycogen during the settling step, the endogenous enzymes must be active and therefore must not be deactivated, for example, by a heating step prior to the settling step.

[0127] Moment of the resting step

[0128] At an acidic pH, the original biomass, lysate, and solubilized material are allowed to stand for at least 3 hours. Specifically, according to the invention, the standing can be performed before or after the cell lysis step b), i.e., the original biomass, lysate, and / or solubilized material are allowed to stand.

[0129] According to the invention, the settling step can advantageously be carried out under stirring of the original biomass and / or pyrolytes and / or solubilizers.

[0130] Preferably, the settling step is performed only on the raw biomass (preferably dried or thawed), pyrolysate, or solubilized material. In the case of pyrolysate and / or solubilized material, if necessary, the settling step is performed after adjusting the pH to obtain the acidic pH required for the settling step.

[0131] Preferably, according to the invention, a settling process is performed after cell lysis step b), in other words, the lysate and / or solubilizer, and even more preferably the lysate, is settling.

[0132] Preferably, the medium containing lysed cells is subjected to a settling step, i.e., the lysate from step b) and / or, where applicable, the solubilizer from step c).

[0133] Conditions of the resting step

[0134] According to the present invention, a settling step may be performed on raw biomass, pyrolysates, and / or solubilizers that are in a dark environment or exposed to natural or artificial light. Preferably, the settling step is performed on raw biomass, pyrolysates, or solubilizers that are in a dark environment.

[0135] According to the present invention, the raw biomass, pyrolysate, or solubilizer is subjected to a settling step with or without ventilation.

[0136] During the settling phase, the “acidic pH” of the glycogen-containing medium is defined as a pH below 7, specifically between 1 and 6, preferably between 2 and 5, and even more preferably between 3 and 4.

[0137] For primitive biomass, glycogen is in the intracellular medium of the cells that make up the biomass, which is a liquid medium with an acidic pH.

[0138] Optionally, the method according to the invention further includes the step of adjusting the pH of the original biomass, pyrolysate and / or solubilizer to the acidic pH of the settling step.

[0139] In this case, the pH of the original biomass, pyrolysate and / or solubilizer is adjusted to a pH below 7, specifically between 1 and 6, even more preferably between 2 and 5, and even more preferably between 3 and 4.

[0140] The reagent used to adjust the pH, i.e., the reagent for acidifying or alkalizing the original biomass, pyrolysate, or solubilization, can be added in solid or solution form. Advantageously, the pH of the original biomass, pyrolysate, or solubilization is adjusted by adding an acidic or alkaline solution, preferably in the form of an aqueous solution.

[0141] Examples of pH-adjusting compounds are given in the description of step c) above: sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, sodium hydroxide, sodium carbonate, or sodium bicarbonate.

[0142] It should be understood that technicians will be able to determine whether acidic or basic compounds or solutions need to be added to the raw biomass, pyrolysate, and / or solubilizer to adjust the pH to the desired value.

[0143] When present, this pH adjustment step is performed upstream of the settling step on the preferably thawed and / or dried raw biomass, on the lysate, or on the solubilizer.

[0144] Advantageously, when present, the pH adjustment step is performed on the lysate before the settling step and before the dilution step c) and the separation step d).

[0145] Alternatively, the pH adjustment step may be performed simultaneously with the dilution step c) (if present). Specifically, if step c) is present, the pH adjustment step may be performed on the lysate simultaneously with the dilution step c) before the settling step. In this case, after diluting the lysate, the obtained solubilized product has a pH below 7, specifically between 1 and 6, preferably between 2 and 5, and even more preferably between 3 and 4.

[0146] The settling step can last up to a week or 7 days. Preferably, the settling step lasts between 3 hours and a week, even more preferably between 3 hours and 48 hours, even more preferably between 6 hours and 36 hours, and even more preferably between 10 hours and 24 hours.

[0147] According to the implementation plan, the settling period lasts from several hours to several days, specifically approximately 3 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, between 48 hours and 72 hours, between 2 days and 7 days, between 3 days and 7 days, 4 days, 5 days, or 6 days.

[0148] According to one embodiment, during the settling phase, the temperature of the original biomass and / or pyrolysate and / or solubilizer is maintained between 15°C and 70°C, specifically between 15°C and 50°C, preferably between 15°C and 40°C, and even more preferably between 15°C and 30°C.

[0149] Advantageously, during this settling stage, the temperature of the original biomass and / or pyrolysate and / or solubilizer is maintained at a constant temperature, specifically between 15°C and 70°C, specifically between 15°C and 50°C, preferably between 15°C and 40°C, and even more preferably between 15°C and 30°C.

[0150] In another embodiment, during the settling phase, the temperature of the original biomass and / or pyrolyte and / or solubilizer is maintained below 15°C, preferably between 4°C and 15°C.

[0151] Advantageously, according to this other embodiment, during the settling phase, the temperature of the original biomass and / or lysate and / or solubilized material is maintained at a constant temperature, specifically between 4°C and 15°C. It should be noted that for the settling step, the closer to the optimal temperature and pH combination, the less time is required for glycogen degradation; for example, for a settling step at a pH between 3 and 4 and a temperature between 15°C and 30°C, the duration can be between 2 hours and 10 hours, while for a settling step at a pH of about 6 and a temperature of about 10°C, the duration should be between at least 6 days and 7 days.

[0152] In the context of this invention, the settling step is performed using a liquid medium containing glycogen, which is free from significant microbial contamination. Such contamination can be avoided by adding a preservative to the liquid medium containing glycogen. These preservatives are well known to those skilled in the art and are particularly 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 EDTA, methylparaben, natamycin, propylparaben, and mixtures thereof.

[0153] Optional addition of exogenous enzymes

[0154] The method may also include the step of adding an exogenous enzyme to supplement the resting step. This addition of an exogenous enzyme enables glycogenolysis to be completed as needed, particularly if the desired level of degradation is not reached. It should be understood that "supplement" here means that the majority (i.e., at least 50%) of the glycogen degradation is a result of the resting step, regardless of this possible addition.

[0155] The term "enzyme" refers to a protein that activates or accelerates a chemical or biological reaction. In the context of this application, the term "exogenous enzyme" refers to an enzyme that is not naturally produced by the cells or microorganisms of treated biomass from single-celled red algae (URA). Specifically, the exogenous enzyme is selected from enzymes extracted from the genera *Aspergillus*, *Bacillus*, or *Trichoderma*.

[0156] The exogenous enzymes according to the invention have glycogen-degrading activity. These enzymes are well known to those skilled in the art and are particularly selected from enzymes having α1-4-glucuronidase activity, α1-4-glucosidase activity, α1-6-glucosidase activity, and amylase activity. It should be noted that any one of these enzymes or a mixture thereof can be used. These enzymes have been found to reduce the size of the glucosinolate chains of glycogen present in the medium, which can then be eliminated along with their degradation byproducts in subsequent stages.

[0157] Enzymes possessing α1-4 glucuronidase activity and / or α1-4 glucosidase activity include pectinases known to degrade pectin, and specifically pectinases extracted from filamentous fungi such as Aspergillus, and more specifically from Aspergillus echinospora, such as those produced by Novozymes under the name Pectinex. ® Enzymes for sale.

[0158] Examples of enzymes with α1-6 glucosidase activity are amylopectinases, known to hydrolyze the α1-6 glucosidic bonds of amylopectin, which are also particularly known to debranch starch. These enzymes are typically derived from bacteria, particularly Bacillus species. Patents US 6,074,854 and US 5,817,498, and application WO2009 / 075682 describe such amylopectinases derived from Bacillus deramificans or Bacillus acidopullulyticus. Commercially available amylopectinases are also known, particularly Promozyme D2 and Novozym 26062 from Novozymes, or Optimax L1000 from DuPont-Genencor.

[0159] Among the known amylases with amylase activity that degrade starch, many are known in the art and described in the literature, specifically in patent applications such as WO 2019 / 036721. Commercially available amylases are known, specifically those using the names "Amylase AG XXL" (from Novozymes) or "Panzym". ® AG XXL (from Eaton).

[0160] Enzymes can be used in pure or enriched form, and optionally as a mixture with one or more excipients. The enzymes used in the methods of the present invention are in powder or solution form. In the latter case, the enzymes are preferentially dissolved in water.

[0161] Preferred conditions for using exogenous enzymes are a pH below 7 and a reaction temperature below 60°C, preferably below 50°C, and even below 30°C. Specifically, the temperature of the solution in which the exogenous enzyme is added and the enzymatic reaction occurs is between 4°C and 60°C, preferably between 20°C and 42°C, and the pH of the solution is less than or equal to 5, preferably about 4.5. The exogenous enzyme can be added to the medium in free form or in a form immobilized on a carrier.

[0162] Exogenous enzymes can be added to the lysate, in which case the lysate is subjected to an enzymatic reaction with the exogenous enzyme, while the original biomass and / or the lysate and / or the lysate are pre-treated by a settling step.

[0163] In a preferred embodiment, the exogenous enzyme is added after the settling step. In this case:

[0164] When the raw biomass undergoes a settling step, exogenous enzymes can be added to the lysate and / or solubilizer and / or clarifying fraction to complete the glycogen degradation resulting from the settling of the raw biomass.

[0165] - When performing a settling step on the lysate, an exogenous enzyme may be added to the lysate after the settling step, or an exogenous enzyme may be added to the solubilizer and / or clarifying fraction to complete the glycogen degradation resulting from the settling of the lysate;

[0166] - When performing a settling step on the solubilized fraction, an exogenous enzyme may be added to the solubilized fraction after the settling step, and / or an exogenous enzyme may be added to the clarified fraction to complete the glycogen degradation resulting from the settling of the solubilized fraction;

[0167] According to one or even a more preferred embodiment, the lysate is subjected to a settling step, and preferably any addition of exogenous enzymes is made to the clarified fraction or solubilized fraction.

[0168] Compared to existing methods, this combination of static incubation and the addition of exogenous enzymes reduces the amount of exogenous enzymes used.

[0169] For enzymes with activity comparable to that of Pectinex Ultra SP-L enzymes (manufacturer-stated activity of 3300 PGNU / g), the proportion of exogenous enzyme added is less than 0.01 by weight relative to the total weight of the raw biomass, lysate, solubilizer, or clarified fraction to be treated.

[0170] In one embodiment, the added exogenous enzyme content is less than or equal to 0.005% by weight, preferably less than or equal to 0.0025% by weight, and more preferably less than or equal to 0.0001% by weight. It should be noted that, if necessary, the concentration of the exogenous enzyme can be adjusted according to the activity of the selected exogenous enzyme to achieve an activity in the reaction medium equivalent to that of 3300 PGNU / g of Pectinex Ultra SP-L according to the present invention.

[0171] Those skilled in the art will also know how to adjust the amount of enzyme to be added during the method to increase the degradation of glycogen present in the lysate, solubilizer, and / or clarified fraction.

[0172] When at least 10%, preferably at least 50%, or even more preferably 50% to 80% of the initial glycogen content in the original biomass has been degraded, the glycogen degradation step is considered sufficient, specifically sufficient to improve subsequent filtration steps, with or without the addition of exogenous enzymes.

[0173] e) Concentration

[0174] The method according to the invention may further include step e) of concentrating the clarified fraction using conventional dehydration methods to obtain a concentrated aqueous extract.

[0175] Conventional water removal methods are known to those skilled in the art, including filtration, evaporation under atmospheric pressure or vacuum, atomization, infrared drying, refracting window drying, and freeze drying.

[0176] Preferably, step e) of the method according to the invention can concentrate the molecules of interest while retaining the basic components of the clarified fraction.

[0177] Depending on the concentration method chosen, concentration step e) specifically removes all or some of the impurities present in the clarified fraction, such as solid residues, residual glycogen, oligomers and sugars produced by glycogen degradation, by means of filtration concentration.

[0178] Even more preferably, the biomass processing method according to the invention includes step e) of concentrating the clarified fraction by filtration (specifically by tangential filtration such as ultrafiltration).

[0179] Preferably, the biomass processing method according to the invention includes a concentration step e), during which the clarified fraction is concentrated by a factor between 2 and 1000, or even more preferably by a factor between 20 and 60.

[0180] Preferably, step e is carried out at a pH less than 7, preferably between 1 and 6, even more preferably between 2 and 5, and even more preferably between 3 and 4.

[0181] In addition to further degradation and / or concentration of the clarified fraction by adding enzymes, the method according to the invention may include subsequent steps for purifying the clarified fraction and / or concentrating the aqueous extract, specifically for purifying proteins in solution.

[0182] Product according to the invention

[0183] The products according to the invention comprise a variety of organic materials, depending on the processing method applied, including water-soluble proteins (including phycocyanin), sugars (including glycogen degradation byproducts (glucose oligomers)) and possible residual undigested glycogen and insoluble matter.

[0184] When present, phycocyanin may include phycocyanins resistant to acidic pH. Phycocyanins resistant to acidic pH are those that are stable at acidic pH, meaning they do not precipitate or discolor at acidic pH. Tolerance or stability to acidic pH can be measured as less than 10% discoloration after exposure to acidic pH (i.e., pH below 7, specifically between pH 2 and 5) for at least 10 minutes. Stability at acidic pH can also be measured by other methods, such as protein structure monitoring.

[0185] According to the extraction method described in WO2018 / 178334, steps d) and optional step e) are performed at pH below 7, preferably between 1 and 6, even more preferably between 2 and 5, and even more preferably between 3 and 4, resulting in the presence of phycocyanin resistant to acidic pH.

[0186] According to a preferred embodiment, the product of the invention comprises phycocyanin resistant to acidic pH.

[0187] Concentrated aqueous extracts are obtained by the method according to the invention, including step e) of the concentrated clarifying fraction as described above. Conversely, clarified fractions are obtained by the method according to the invention, excluding step e) of the concentrated clarifying fraction as described above.

[0188] Preferably, the product according to the invention does not contain exogenous enzymes or contains a concentrate of exogenous enzymes that are undetectable by conventional assay methods (especially after protein precipitation with acetonitrile, digestion with trypsin, and then analysis by mass spectrometry (LC-MS-MS)).

[0189] Specifically, the products according to the invention preferably do not contain exogenous enzymes selected from pectinase, amylase and amylopectinase, or contain concentrates of exogenous enzymes that are undetectable by conventional assay methods (specifically mass spectrometry).

[0190] If desired, the product according to the invention is prepared in a manner that eliminates any impurities that would make it unsuitable for consumption (specifically, unsuitable for human consumption).

[0191] The products according to the invention can also be formulated by methods known to those skilled in the art to avoid degradation of their components during storage or subsequent use.

[0192] Finally, the product according to the invention (which may be a formulated product) may be packaged for storage and use, either in a large-volume container or in a smaller container, for example having a volume corresponding to a single use, referred to as a single dose, for human consumption. In this case, the container may be rigid, such as a glass ampoule, or flexible, such as a capsule suitable for consumption.

[0193] Following acid hydrolysis of the sample, the sugar content of the product according to the invention, specifically glucose, mannose, and galactose (referred to as "hydrolyzed glucose," "hydrolyzed galactose," and "hydrolyzed mannose," respectively), was measured using high-performance liquid chromatography with a Hi-Plex H+ ion exclusion / ligand exchange column and refractive index detection (hereinafter referred to as "HPLC-RID determination of hydrolyzed sugars"). For this purpose, 1.5 mL of the supernatant from the vortex-homogenized sample was hydrolyzed with 1.5 mL of 2N sulfuric acid at 110 °C for 2 hours. The sample was then filtered (0.22 µm) and analyzed.

[0194] It should be noted that the hydrolyzed glucose content measured in this way includes both undigested residual glycogen glucose and free glucose present in the product.

[0195] According to the present invention, the free glucose content is determined by biochemical analysis, specifically using YSI. ® The biochemical analyzer was used according to the manufacturer's recommendations.

[0196] Therefore, in order to estimate the content of undigested residual glycogen in the product according to the invention, the content of free glucose is simply subtracted from the content of hydrolyzed glucose.

[0197] The percentage of glycogen degradation is calculated as follows: (concentration of free glucose in the studied sample / maximum concentration of free glucose in an equivalent sample treated with 1% exogenous enzyme relative to the total sample volume) × 100.

[0198] According to the present invention, the protein content of the product is determined by the DUMAS method. The sample is subjected to high-temperature combustion in a stream of pure oxygen, and the resulting nitrogen oxides are reduced with copper. After separation of reaction byproducts, nitrogen is measured using a thermal conductivity detector, and the result is expressed as N (%). This percentage of nitrogen is then converted to the amount of protein by applying the following formula: N 6.25 (%) (ISO / TS 16634-2:2009).

[0199] Finally, to determine the C-phycocyanin content of the sample according to the present invention, 500 μL of sample was mixed with 100 mM Tris-CI buffer (pH 7.5, 1.5 ml), and the absorbance at 652 nm and 620 nm was measured using a Metier Toledo spectrophotometer. The C-phycocyanin concentration was then calculated using the following formula:

[0200]

[0201] According to the present invention, glycogen degradation of less than 10% by weight of total glycogen is not considered sufficient degradation. Furthermore, degradation of less than 10% does not improve extract filtration. Therefore, glycogen degradation is considered ineffective.

[0202] According to one embodiment, the clarified fraction 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.

[0203] The clarified fraction according to the present invention has a total sugar concentration of less than or equal to 40 g / L, specifically between 0.1 g / L and 40 g / L, preferably between 1 g / L and 20 g / L, and more preferably between 3 g / L and 11 g / L, as measured by HPLC-RID determination of hydrolyzed sugar.

[0204] And / or less than or equal to 20 g / L, preferably between 0.1 g / L and 20 g / L, preferably between 1 g / L and 10 g / L, and even more preferably between 2 g / L and 5 g / L of hydrolyzed glucose concentration.

[0205] And / or less than or equal to 10 g / L, specifically between 0.01 g / L and 10 g / L, preferably between 0.1 g / L and 5 g / L, more preferably between 0.5 g / L and 3 g / L, of hydrolyzed galactose concentration.

[0206] And / or less than or equal to 10 g / L, specifically between 0.01 g / L and 10 g / L, preferably between 0.1 g / L and 5 g / L, more preferably between 0.5 g / L and 3 g / L, hydrolyzed mannose concentration.

[0207] And / or a free glucose concentration less than or equal to 10 g / L, specifically between 0.01 g / L and 10 g / L, preferably between 0.1 g / L and 5 g / L, and even more preferably between 0.2 g / L and 2 g / L.

[0208] The C-phycocyanin content of the clarified fraction is advantageously in the range of 0.1 g / L to 12 g / L, preferably 0.5 g / L to 8 g / L, and more preferably 1 g / L to 7 g / L.

[0209] Preferably, the clarified fraction has a ratio of at least 0.001, preferably 0.005 to 120, more preferably 0.05 to 12, and even more preferably 0.1 to 3 (C-phycocyanin (g / L) / total sugar (g / L) as measured by HPLC-RID determination of hydrolyzed sugar).

[0210] According to another preferred embodiment of the invention, the clarified fraction comprises C-phycocyanin and total sugar, wherein the ratio of the two (C-phycocyanin (g / L) / total sugar (g / L) as measured by HPLC-RID determination of hydrolyzed sugar) is 0.005 to 120, specifically 0.05 to 12, more preferably 0.1 to 3.

[0211] In a specific embodiment of the concentrated aqueous extract having a high concentration of C-phycocyanin, the ratio (C-phycocyanin (g / L) / total sugar (g / L)) is at least 90 and may exceed 120.

[0212] Preferably, the clarified fraction has a ratio of at least 0.01, preferably 0.01 to 1200, more preferably 0.1 to 80, or even more preferably 0.1 to 12 (C-phycocyanin (g / L) / hydrolyzed mannose (g / L)).

[0213] Preferably, the clarified fraction has a ratio of at least 0.01, preferably 0.01 to 1200, more preferably 0.1 to 80, or even more preferably 0.1 to 12 (C-phycocyanin (g / L) / hydrolyzed galactose (g / L)).

[0214] The ratio of the clarified fractions (C-phycocyanin (g / L) / hydrolyzed glucose (g / L)) is preferably less than 120, and preferably between 0.005 and 120. Preferably, the ratio is between 0.05 and 12, and more preferably between 0.1 and 10.

[0215] Preferably, the concentrated aqueous extract according to the invention has a dry matter content of 2 to 1000 times, or even more preferably 20 to 60 times, that of the clarified fraction from which it is derived.

[0216] The C-phycocyanin content of the concentrated aqueous extract is advantageously in the range of 7 g / L to 120 g / L, preferably 20 g / L to 100 g / L, and more preferably 40 g / L to 80 g / L.

[0217] The concentrated aqueous extract according to the invention has a total sugar concentration of less than or equal to 50 g / L, specifically between 1 g / L and 50 g / L, preferably between 5 g / L and 40 g / L, and more preferably between 10 g / L and 30 g / L, as measured by HPLC-RID determination of hydrolyzed sugar.

[0218] And / or less than or equal to 20 g / L, preferably between 0.1 g / L and 20 g / L, preferably between 1 g / L and 10 g / L, and even more preferably between 2 g / L and 5 g / L of hydrolyzed glucose concentration.

[0219] And / or less than or equal to 15 g / L, specifically between 0.5 g / L and 15 g / L, preferably between 1 g / L and 13 g / L, more preferably between 3 g / L and 8 g / L of hydrolyzed galactose concentration.

[0220] And / or less than or equal to 15 g / L, specifically between 0.5 g / L and 15 g / L, preferably between 1 g / L and 13 g / L, more preferably between 3 g / L and 8 g / L of hydrolyzed mannose concentration.

[0221] And / or less than or equal to 15 g / L, specifically between 0.01 g / L and 15 g / L, preferably between 0.5 g / L and 8 g / L, and even more preferably between 1 g / L and 4 g / L of free glucose concentration.

[0222] The weight ratio of the concentrated aqueous extract (C-phycocyanin (g / L) / hydrolyzed glucose (g / L)) is in the range of 2 to 80. Preferably, the ratio is between 10 and 70, more preferably between 15 and 60.

[0223] Preferably, the concentrated aqueous extract has a ratio of at least 0.01, preferably 0.01 to 1200, more preferably 0.1 to 80, and even more preferably 0.3 to 12 (C-phycocyanin (g / L) / hydrolyzed mannose (g / L)).

[0224] Preferably, the concentrated aqueous extract has a ratio of at least 0.01, preferably from 0.01 to 1200, more preferably from 0.1 to 80, and even more preferably from 0.3 to 12 (C-phycocyanin (g / L) / hydrolyzed galactose (g / L)).

[0225] Preferably, the concentrated aqueous extract has a ratio of 0.2 to 6.0, more preferably 0.5 to 5.0, and even more preferably 1.0 to 4.5 (protein (%) / estimated undigested residual glycogen (g / L)).

[0226] Preferably, the concentrated aqueous extract has an estimated undigested residual glycogen content (hydrolyzed glucose content (g / L) - free glucose content (g / L)) of 0.1 g / L to 10 g / L, more preferably 0.2 g / L to 7 g / L, and even more preferably 0.3 g / L to 5 g / L.

[0227] Preferably, the concentrated aqueous extract has a ratio of less than 1%, preferably between 1% and 0.01%, more preferably between 0.8% and 0.05%, and even more preferably between 0.6% and 0.1% (protein (%) / total sugar (g / L) as measured by HPLC-RID determination of hydrolyzed sugar).

[0228] Finally, the present invention relates to a method for preparing a concentrated aqueous extract of microalgal ARU biomass with high glycogen content that produces phycocyanin, the method comprising a treatment method having a glycogen degradation step and a concentration step as previously defined.

[0229] Use

[0230] This invention also relates to the use of all or part of the product obtained according to the method of the invention as a product for the preparation of pharmaceutical or dietary compositions, including food or pharmaceuticals, nutritional products, diets, cosmetics, or industrial compositions. Specifically, the use of the product containing phycocyanin obtained according to the method of the invention for the preparation of pharmaceutical or dietary compositions.

[0231] According to one embodiment, the composition according to the invention comprises all or part of the product obtained by the method according to the invention and one or more excipients.

[0232] According to a preferred embodiment, the composition according to the invention is an acidic composition having a pH less than or equal to 6, preferably less than or equal to 5, even less than or equal to 4, and specifically between 2 and 4.

[0233] According to the present invention, an acidic composition means any composition comprising all or part of the product obtained by the method according to the present invention, and an inorganic or organic acid.

[0234] Inorganic or organic acids that can be used in the compositions according to the present invention are known to those skilled in the art. Inorganic acids include carbonic acid, phosphoric acid, hydrochloric acid, sulfuric acid, perchloric acid, sulfonic acid, and nitric acid. Organic acids include citric acid, lactic acid, malic acid, tartaric acid, and succinic acid.

[0235] The acidic compositions according to the invention may further comprise a mediator, which may contain structural components associated with an active compound identified based on its nutritional contribution or its properties beneficial to human or animal health.

[0236] Compositions other than dietary compositions may be pharmaceuticals, veterinary drugs, or cosmetics, and may also contain one or more additives and / or active ingredients known for use in such indications.

[0237] The compositions according to the invention can exist in any commonly known form. These forms include solid, liquid, fluid, paste, or viscous forms, specifically creams, gels, foams, and pastes. The compositions according to the invention can also be in the form of dry foods for cooking, powders for dilution, or gel compositions for food compositions.

[0238] In these solid compositions, all or part of the extract obtained according to the invention is preferably added in powder form. In this case, the product obtained by the method according to the invention is first dried to present it in powder form.

[0239] The liquid composition is advantageously an aqueous composition in which all or part of the extract obtained according to the invention is dissolved.

[0240] According to one specific embodiment of the invention, the liquid composition may be a dietary composition, and more specifically, an acidic, carbonated, or non-carbonated beverage. Examples include soda water, fruit juice, sports drinks, functional drinks, energy drinks, etc. The compositions of these beverages are well known to those skilled in the art and may contain sugars, mineral salts, food additives, or dissolved gases. The acidic beverage according to the invention may be a prior art acidic beverage in which commonly used dyes are wholly or partially replaced by a product containing phycocyanin resistant to acidic pH according to the invention.

[0241] The phycocyanin content in the composition according to the present invention will be consistent with the concentration commonly used in the intended application field.

[0242] Examples

[0243] Example 1 : Analysis of glycogen degradation with and without addition of exogenous enzymes at different pH levels

[0244] The amount of free glucose in *Galdirius sulfadiazine* biomass lysate over time was measured as a function of time (YSI) at 37°C, pH 3.75 without enzyme, pH 6 without enzyme, and pH 6 with enzyme (enzyme solution volume 1% of total lysate volume, enzyme being Novozymes' "Amylase AG XXL"). ® 2950).

[0245] These dynamics are shown in Figure 1 middle.

[0246] The lysate was acidified by adding sufficient citric acid to change the pH from 6 to 3.75.

[0247] The results showed that more glucose was released at the acidic pH of 3.75 than at pH 6. After incubation at pH 3.75 without enzyme addition for 24 hours, the free glucose in the sample even reached the same level as in the sample with added exogenous enzyme.

[0248] It should be noted that glucose release also occurs at pH 6 in the absence of exogenous enzymes; however, the kinetics are slower (the slope is gentler).

[0249] Example 2: Analysis of glycogen degradation without addition of exogenous enzymes at different pH levels

[0250] The amount of free glucose (YSI) over time in *Galdirius sulfadiazine* biomass lysates (20% by weight dry matter (DM) of the total lysate weight) with pH between 3 and 7 was measured without the addition of enzymes and at room temperature (20°C). ® 2950).

[0251] These dynamics are shown in Figure 2 middle.

[0252] The acidification and alkalization of the pyrolysis products are achieved by adding sufficient amounts of citric acid or sodium hydroxide, respectively.

[0253] The results showed that, within a given time period, higher pH values ​​resulted in less glycogen degradation. Glycogen digestion in the lysate was active under the most acidic conditions without the addition of exogenous enzymes. This activity was observed to be higher at pH 3 but slightly lower at pH 4. Activity was similar at pH levels 5 through 7, but slower than at lower pH levels.

[0254] Example 3: Analysis of glycogen degradation with and without addition of exogenous enzymes at different temperatures

[0255] The amount of free glucose (YSI) in *Galdirius thiophanate-methyl* biomass lysate (20% by weight dry matter (DM) of total lysate weight) at pH 3.75 was analyzed over time at different temperatures (4°C, 20°C, and 37°C) under conditions with and without enzyme (1% "Amylase AGXXL" from Novozymes). ® 2950).

[0256] The results are presented in Figure 3 The results showed that even without the addition of enzymes, glycogen was digested into free glucose. Specifically, after 24 hours at 37°C, the amount of free glucose was the same with and without the addition of enzymes.

[0257] Example 4: Analysis of glycogen degradation without addition of exogenous enzymes at different temperatures

[0258] Analysis of the amount of free glucose (YSI) over time in *Galdirius sulfadiazine* biomass lysate (20% by weight dry matter (DM) of total lysate weight) at different temperatures (4℃, 15℃, 20℃, 30℃, 40℃, and 50℃) and pH 4 without the addition of exogenous enzymes. ® 2950).

[0259] The results are presented in Figure 4 The results showed that, within a given time period, the higher the temperature, the greater the amount of glycogen digested during the settling step according to the invention.

[0260] Example 5: Analysis of glycogen degradation at different stages of the extraction process

[0261] The amount of free glucose (YSI) in the lysate (20% wt dry matter (DM) relative to the total weight of the lysate) and the solubilized galdiridia biomass (7.5% wt DM relative to the total weight of the solubilized matter) at pH 3.75 at room temperature (20°C) without the addition of exogenous enzymes was analyzed. ® 2950). In this context, the kinetics of glucose release at room temperature (20°C) in thiophyid algae biomass lysate at pH 3.75 was used as a control with the addition of an exogenous enzyme (enzyme solution volume relative to total lysate volume, enzyme being “Amylase AG XXL” from Novozymes).

[0262] These dynamics are shown in Figure 5 middle.

[0263] From the results obtained and by extrapolation, it can be concluded that even without the addition of enzymes, in the case of the lysate, all digestible glycogen will be degraded and released as free glucose after 24 hours. On the other hand, in the case of the solubilizer, the reaction appears to be slower and still incomplete after 24 hours.

[0264] It should be noted that all the samples studied showed more than 10% glycogen degradation, thus exhibiting good filterability after glycogen digestion, regardless of whether they were prepared with or without the addition of exogenous enzymes.

[0265] Example 6: Analysis of glycogen degradation in the prior art

[0266] In patent WO2020 / 144330, Example 2 illustrates glycogen degradation in crude phycocyanin solution with and without exogenous enzymes.

[0267] To achieve this, sulfur-loving Galdiri algae biomass is produced and then recycled / harvested. The cells are then lysed and diluted to obtain a dissolved aqueous extract. The solids are then separated by filtration through a 0.22µm filter to obtain a clarified fraction from this aqueous extract.

[0268] The amount of free glucose in the *Galdiridia sulfadiazine* biomass clarified fraction (7.5 wt% MS, relative to the total clarified fraction volume) at pH 4 was analyzed at room temperature (20°C) without and with the addition of exogenous enzymes (enzyme solution volume relative to the total clarified fraction volume, enzymes being "Amylase AG XXL", "Pectinex Ultra SP-L", or "BAN 480 L" from Novozymes). (YSI 2700 Biochemical Analyzer)

[0269] The results are presented in Figure 6 middle.

[0270] The results show that without the addition of exogenous enzymes and with the clarified fraction left to stand for 95 hours, glycogen was not fully digested (degradation less than 10%). However, with the addition of enzymes, all glycogen was digested after 95 hours.

[0271] Example 7: Analysis of glycogen degradation at all stages of the extraction process

[0272] The study analyzed the amount of free glucose (YSI) in the lysate (20% wt DM relative to the total weight of the lysate), solubilized fraction, and clarified fraction of *Galdirius thiophilus* biomass (7.5% wt DM relative to the total weight of the solubilized fraction or clarified fraction) at pH 4 at room temperature (20°C) without the addition of exogenous enzymes. ® 2950). The clarified fraction was obtained after filtering the solubilizer through a 0.22µm filter.

[0273] In this context, the maximum free glucose concentration in the samples was determined using glucose release kinetics in the lysate, solubilized fraction, and clarification fraction of *Galdirius thiophilus* biomass at pH 4 with the addition of an exogenous enzyme (enzyme solution volume relative to total lysate volume, enzyme being "Amylase AG XXL" from Novozymes) at room temperature (20°C).

[0274] These data are used for calculation Figure 7 The percentage of glycogen degradation (digestion) shown is displayed.

[0275] The results show that, without the addition of exogenous enzymes, more than 10% of the glycogen was digested after 48 hours when the lysate and solubilized fraction were left to stand, but the clarified fraction was not digested.

[0276] Example 8: Characterization of the extract prepared according to the invention

[0277] The pyrolysis product of *Galdirius sulfadiazine* (20% by weight of DM of the total pyrolysis product) was kept at 25°C and at an acidic pH of 3.7 for 12 hours.

[0278] An extract was prepared from the pyrolysis product. To do this, the pyrolysis product was washed with a volume of water less than four times the total volume of the pyrolyzed biomass. This volume of water was divided into three portions for three consecutive washes of the pyrolyzed biomass. The wash water was recovered according to the teachings of patent application WO2020 / 161280. The resulting product was then filtered through a hollow fiber membrane with a porosity of 70 kDa (clarification fraction) via a final percolation step according to the teachings of patent application WO2020 / 144330, and the filtrate (concentrated aqueous extract) was recovered.

[0279] Total sugars, hydrolyzed mannose, hydrolyzed galactose, and hydrolyzed glucose in the aqueous extract concentrate and clarified fraction were analyzed by HPLC-RID, and YSI was used. ® 2950. Free glucose was analyzed, along with phycocyanin and other proteins.

[0280] The results are presented in Table 1 below.

[0281] Table 1

[0282]

Claims

1. A method for processing biomass of single-celled red algae (URA) of the genus *Gardilia*, the URA biomass processing method comprising the following steps: a) Harvesting biomass through a separation medium to obtain the original biomass for URA. b) The crude biomass from step (a) is subjected to cell lysis to obtain lysate. c) Optionally, the lysate from step (b) can be diluted to obtain a solubilizer, and d) Separate the insoluble fractions suspended in the pyrolysate from step (b) or the solubilizer from step (c) to obtain a clarified fraction. The method is characterized by comprising a glycogen degradation step, wherein the original biomass and / or pyrolytes and / or, if applicable, solubilizers are kept in a static state (static step) for at least 3 hours, during which time the liquid medium containing glycogen is at an acidic pH.

2. The method according to claim 1, characterized in that, The settling process lasts between 3 hours and one week.

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

4. The method according to any one of claims 1 to 3, characterized in that, The settling step is preferably performed on the pyrolysis product and / or, if applicable, the solubilizer.

5. The method according to any one of claims 1 to 4, characterized in that, The method includes, prior to the settling step, adjusting the pH of the original biomass, pyrolyte, and / or, if applicable, solubilizer to below 7, specifically between 1 and 6, preferably between 2 and 5, and more preferably between 3 and 4.

6. The method according to any one of claims 1 to 5, characterized in that, The method further includes the step e) of concentrating the clarified fraction to obtain a concentrated aqueous extract.

7. A product that can be obtained by the method according to any one of claims 1 to 6.

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

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

10. The product according to any one of claims 7 or 9, characterized in that, The product contains a concentration of hydrolyzed galactose less than or equal to 15 g / L.

11. The product according to any one of claims 7 to 10, characterized in that, The product comprises a protein (%) ratio of 0.2 to 6.0, more preferably 0.5 to 5.0, and even more preferably 1.0 to 4.5% as estimated by HPLC-RID determination of hydrolyzed sugars (g / L).

12. The product according to any one of claims 7 to 11, characterized in that, The product contains less than 1%, preferably between 1% and 0.01%, more preferably between 0.8% and 0.05%, and even more preferably between 0.6% and 0.1% of protein (%) / total sugar (g / L) as measured by HPLC-RID determination of hydrolyzed sugar.

13. The product according to any one of claims 7 to 12, characterized in that, The product contains between 0.1 g / L and 20 g / L of C-phycocyanin.

14. A nutritional, dietary, or cosmetic composition comprising the product according to any one of claims 7 to 13.

15. The composition according to claim 14, characterized in that, The composition contains inorganic or organic acids.

Images